Devices and methods for generation and culture of 3d cell aggregates

ABSTRACT

The present disclosure relates to apparatuses, systems and methods for culturing cells. In particular, devices and methods are provided for generation and culture of 3d cell aggregates.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation application of International Patent ApplicationSerial No. PCT/US15/58048 filed on Oct. 29, 2015, which claims thebenefit of priority to U.S. Provisional Application Ser. No. 62/072,015,filed on Oct. 29, 2014, the contents of which are relied upon andincorporated herein by reference in their entirety, and the benefit ofpriority under 35 U.S.C. §120 is hereby claimed.

FIELD

The present disclosure relates to apparatus, systems and methods forculturing cells. In particular, devices and methods are provided forgeneration and culture of 3d cell aggregates.

BACKGROUND

Three dimensional (3D) cell culture is the growth of cells in anartificially-created environment that allows the cells to grow and/orinteract primarily with each other in all three dimensions. 3D cellculture represents an improvement over methods of growing cells in 2D(e.g., on a petri dish) for at least the reason that the 3D conditionsmore accurately model the in vivo environment.

Cells cultured in three dimensions, such as spheroids, can exhibit morein-vivo like functionality than their counterparts cultured in twodimensions as monolayers. In two dimensional cell culture systems, cellscan attach to a substrate on which they are cultured. However, whencells are grown in three dimensions, such as spheroids, the cellsinteract with each other rather than attaching to the substrate. Oneissue with spheroid based assays is that the assay results typicallyvary with the size of the spheroid. For example, variations invariables, such as seeding density and growth time, from system tosystem may affect assay repeatability from system to system or from wellto well within a given system. Accordingly, maintaining a consistentspheroid size between spheroids grown in separate wells can presentchallenges.

As the density of cells grown in cell culture apparatus increases,larger volumes of cell culture media or more frequent exchange of cellculture media may be needed to maintain the cells. However, increasedfrequency of media exchange can be inconvenient. In addition, increasedvolumes of cell culture media can lead to undesirably increased heightof media above the cultured cells. As the media height increases, gasexchange rate for the cells through the media decreases.

Cells have been grown in high density as spheroid clusters in wave bags,spinners and shakers. However, the size of spheroids grown in suchapparatuses is inconsistent and the shear inherent in such apparatusestends to break spheroids into smaller clusters. Further, suchapparatuses may not been able to achieve cell densities sufficientlyhigh to satisfy current demands.

SUMMARY

The present disclosure relates to apparatus, systems and methods forculturing cells. In particular, devices and methods are provided forgeneration and culture of 3d cell aggregates. For example, devices andmethods are provided to address issues known or unknown to the field tobe detrimental to the 3D culturing of cells.

Cells cultured in three dimensions, such as spheroids, can exhibit morein vivo like functionality than their counterparts cultured in twodimensions as monolayers. In two dimensional cell culture systems, cellscan attach to a substrate on which they are cultured. However, whencells are grown in three dimensions, such as spheroids, the cellsinteract with each other rather than attaching to the substrate. Cellscultured in three dimensions more closely resemble in vivo tissue interms of cellular communication and the development of extracellularmatrices. Spheroids thus provide a superior model for cell migration,differentiation, survival, and growth and therefore provide bettersystems for research, diagnostics, and drug efficacy, pharmacology, andtoxicity testing.

In some embodiments, a substrate is provided that contains or comprisesan array of microwells or wells. The substrate can form a part of a cellculture apparatus or device. For example, the substrate can form a partof a multiwell plate, a flask, a dish, a tube, a multi-layer cellculture flask, a bioreactor, or any other laboratory container intendedto grow cells or spheroids. The microwells or wells (the term“microwell” and “well” are used interchangeably in this disclosure) arestructured and arranged to provide an environment that is conducive tothe formation of spheroids in culture. That is, in embodiments, themicrowells have spheroid-inducing geometry. In addition, the wells arestructured and arranged to provide for the movement of liquid into andout of the wells without trapping air between the substrate and liquidor liquid droplets that are introduced into the wells That is, inembodiments, the microwells have capillary structures. For example, thewells in which cells are grown can be non-adherent to cells to cause thecells in the wells to associate with each other and form spheres. Thespheroids expand to size limits imposed by the geometry of the wells. Insome embodiments, the wells are coated with an ultra-low bindingmaterial to make the wells non-adherent to cells.

In some embodiments, the cell culture devices have frames comprising thefootprint of the device, the substrate of which is configured such thatcells cultured in the devices form spheroids. For example, the cellculture substrate in the devices is non-adherent to cells to cause thecells to associate with each other instead of the substrate. The cellculture substrate is further comprised of a plurality of microwells (orwells), the geometry of which enable cells grown in the wells to formsimilar-sized cell aggregates or spheroids. The spheroids expand to sizelimits imposed by the geometry of the microwells. In some embodiments,the wells are have a low-binding treatment or are coated with anultra-low binding material to make the wells non-adherent to cells.

Examples of non-adherent material include perfluorinated polymers,olefins, or like polymers or mixtures thereof. Other examples includeagarose, non-ionic hydrogels such as polyacrylamides, polyethers such aspolyethylene oxide and polyols such as polyvinyl alcohol, or likematerials or mixtures thereof. The combination of, for example,non-adherent wells, well geometry (e.g., size and shape), and/or gravityinduce cells cultured in the wells to self-assemble into spheroids. Somespheroids maintain differentiated cell function indicative of a more invivo-like, response relative to cells grown in a monolayer. Other cellstypes, such as mesenchymal stromal cells, when cultured as spheroidsretain their pluripotency,

In some embodiments, the systems, devices, and methods herein compriseone or more cells. In some embodiments, the cells are cryopreserved. Insome embodiments, the cells are in three dimensional culture. In somesuch embodiments, the systems, devices, and methods comprise one or morespheroids. In some embodiments, one or more of the cells are activelydividing. In some embodiments, the systems, devices, and methodscomprise culture media (e.g., comprising nutrients (e.g., proteins,peptides, amino acids), energy (e.g., carbohydrates), essential metalsand minerals (e.g., calcium, magnesium, iron, phosphates, sulphates),buffering agents (e.g., phosphates, acetates), indicators for pH change(e.g., phenol red, bromo-cresol purple), selective agents (e.g.,chemicals, antimicrobial agents), etc.). In some embodiments, one ormore test compounds (e.g., drug) are included in the systems, devices,and methods.

A wide variety of cell types may be cultured. In some embodiments, aspheroid contains a single cell type. In some embodiments, a spheroidcontains more than one cell type. In some embodiments, where more thanone spheroid is grown, each spheroid is of the same type, while in otherembodiments, two or more different types of spheroids are grown. Cellsgrown in spheroids may be natural cells or altered cells (e.g., cellcomprising one or more non-natural genetic alterations). In someembodiments, the cell is a somatic cell. In some embodiments, the cellis a stem cell or progenitor cell (e.g., embryonic stem cell, inducedpluripotent stem cell) in any desired state of differentiation (e.g.,pluripotent, multi-potent, fate determined, immortalized, etc.). In someembodiments, the cell is a disease cell or disease model cell. Forexample, in some embodiments, the spheroid comprises one or more typesof cancer cells or cells that can be induced into a hyper-proliferativestate (e.g., transformed cells). Cells may be from or derived from anydesired tissue or organ type, including but not limited to, adrenal,bladder, blood vessel, bone, bone marrow, brain, cartilage, cervical,corneal, endometrial, esophageal, gastrointestinal, immune system (e.g.,T lymphocytes, B lymphocytes, leukocytes, macrophages, and dendriticcells), liver, lung, lymphatic, muscle (e.g., cardiac muscle), neural,ovarian, pancreatic (e.g., islet cells), pituitary, prostate, renal,salivary, skin, tendon, testicular, and thyroid. In some embodiments,the cells are mammalian cells (e.g., human, mice, rat, rabbit, dog, cat,cow, pig, chicken, goat, horse, etc.).

The cultured cells find use in a wide variety of research, diagnostic,drug screening and testing, therapeutic, and industrial applications.

In some embodiments, the cells are used for production of proteins orviruses. Systems, devices, and methods that culture large numbers ofspheroids in parallel are particularly effective for protein production.Three-dimensional culture allows for increased cell density, and higherprotein yield per square centimeter of cell growth surface area. Anydesired protein or viruses for vaccine production may be grown in thecells and isolated or purified for use as desired. In some embodiments,the protein is a native protein to the cells. In some embodiments, theprotein is non-native. In some embodiments, the protein is expressedrecombinantly. Preferably, the protein is overexpressed using anon-native promoter. The protein may be expressed as a fusion protein.In some embodiments, a purification or detection tag is expressed as afusion partner to a protein of interest to facilitate its purificationand/or detection. In some embodiments, fusions are expressed with acleavable linker to allow separation of the fusion partners afterpurification.

In some embodiments, the protein is a therapeutic protein. Such proteinsinclude, but are not limited to, proteins and peptides that replace aprotein that is deficient or abnormal (e.g., insulin), augment anexisting pathway (e.g., inhibitors or agonists), provide a novelfunction or activity, interfere with a molecule or organism, or deliverother compounds or proteins (e.g., radionuclides, cytotoxic drugs,effector proteins, etc.). In some embodiments, the protein is animmunoglobulin such as an antibody (e.g., monoclonal antibody) of anytype (e.g., humanized, bi-specific, multi-specific, etc.). Therapeuticprotein categories include, but are not limited to, antibody-baseddrugs, Fc fusion proteins, anticoagulants, antigens, blood factor, bonemorphogenetic proteins, engineered protein scaffolds, enzymes, growthfactors, hormones, interferons, interleukins, and thrombolytics.Therapeutic proteins may be used to prevent or treat cancers, immunedisorders, metabolic disorders, inherited genetic disorders, infections,and other diseases and conditions.

In some embodiments, the protein is a diagnostic protein. Diagnosticproteins include, but are not limited to, antibodies, affinity bindingpartners (e.g., receptor-binding ligands), inhibitors, antagonists, andthe like. In some embodiments, the diagnostic protein is expressed withor is a detectable moiety (e.g., fluorescent moiety, luminescent moiety(e.g., luciferase), colorimetric moiety, etc.).

In some embodiments, the protein is an industrial protein. Industrialproteins include, but are not limited to, food components, industrialenzymes, agricultural proteins, analytical enzymes, etc.

In some embodiments, the cells are used drug discovery,characterization, efficacy testing, and toxicity testing. Such testingincludes, but is not limited to, pharmacological effect assessment,carcinogenicity assessment, medical imaging agent characteristicassessment, half-life assessment, radiation safety assessment,genotoxicity testing, immunotoxicity testing, reproductive anddevelopmental testing, drug interaction assessment, dose assessment,adsorption assessment, disposition assessment, metabolism assessment,elimination studies, etc. Specific cells types may be employed forspecific tests (e.g., hepatocytes for liver toxicity, renal proximaltubule epithelial cells for nephrotoxicity, vascular endothelial cellsfor vascular toxicity, neuronal and glial cells for neurotoxicity,cardiomyocytes for cardiotoxicity, skeletal myocytes for rhabdomyolysis,etc.). Treated cells may be assessed for any number of desiredparameters including, but not limited to, membrane integrity, cellularmetabolite content, mitochondrial functions, lysosomal functions,apoptosis, genetic alterations, gene expression differences, and thelike.

In some embodiments, the cell culture devices are a component of alarger system. In some embodiments, the system comprises a plurality(e.g., 2, 3, 4, 5, . . . , 10, . . . , 20, . . . , 50, . . . , 100, . .. , 1000, etc.) of such cell culture devices. In some embodiments, thesystem comprises an incubator for maintaining the culture devices atoptimal culture conditions (e.g., temperature, atmosphere, humidity,etc.). In some embodiments, the system comprises detectors for imagingor otherwise analyzing cells. Such detectors include, but are notlimited to, fluorimeters, luminometers, cameras, microscopes, platereaders (e.g., PERKIN ELMER ENVISION plate reader; PERKIN ELMER VIEWLUXplate reader), cell analyzers (e.g., GE IN Cell Analyzer 2000 and 2200;THERMO/CELLOMICS CELLNSIGHT High Content Screening Platform), andconfocal imaging systems (e.g., PERKIN ELMER OPERAPHENIX high throughputcontent screening system; GE INCELL 6000 Cell Imaging System). In someembodiments, the system comprises perfusion systems or other componentsfor supplying, re-supplying, and circulating culture media or othercomponents to cultured cells. In some embodiments, the system comprisesrobotic components (e.g., pipettes, arms, plate movers, etc.) forautomating the handing, use, and/or analysis of culture devices.

In the handling of microwell format microplates or other vessels withmicrowells, and in particular during the addition of liquid to themicrowells, care must be taken to ensure the complete displacement ofair from the microwells upon introduction of aqueous liquid. Uponaddition of liquid to the vessel containing wells, air may be trappedbeneath the liquid but within the well (e.g., microwell), particularlyif the wells have a circular cross section. The surface tension of theaqueous liquid added to the well is strong, such that drops tend toremain spherical. A spherical drop can easily block a circular hole ofsimilar size, causing air trapping within the hole (e.g., themicrowell).

In some embodiments, well geometries are provided herein that reduce thelikelihood that air will become trapped in the microwell, whilemaintaining the cell culture characteristics of the well (e.g., utilityin 3D cell culture). In some embodiments, well geometries (e.g.,microwell geometries) allow for the efficient displacement of air uponintroduction of liquid to the well. In some embodiments, well geometriesprovide pathways for the flow of liquid into the well without blockingthe escape of air from the well. In some embodiments, well geometriesprovide pathways for trapped air to escape. In some embodiments,provided herein are a variety of well geometries that facilitate airdisplacement in microwells and permit the entrance of liquid into themicrowells, while maintaining confinement dimensions for cellaggregation.

In embodiments, the disclosure provides devices (e.g., multiwell plates,petri dishes, flasks, multi-layer flasks, or HyperStacks) for culturingand assaying, for example, spheroidal cell masses or other aggregatecell colonies. In some embodiments, devices comprise at least onechamber (e.g., well (e.g., macrowell), flask, etc.) comprising anopening (e.g., aperture), a side wall or plurality of side walls, and abottom surface having one or more microwells. In some embodiments, thegeometry of the opening, the side wall(s), and the bottom allow for: 3Dculturing of cells (e.g., cell aggregates, spheroids, etc.) within thechamber, as well as one or more (e.g., all) of: (1) displacement of airfrom the chamber upon dispensing of reagents (e.g., liquid reagents)into the chamber (e.g., without air becoming trapped beneath or withinthe liquid), (2) routes for flow of liquid into the microwell thatreduce the likelihood of trapping air beneath the surface of the liquidin the microwell, (3) pathways for the escape of air upon introductionof liquid to the microwell, and/or (4) pathways for the escape of airtrapped beneath the liquid surface.

In some embodiments, devices are prepared with a surface comprising oneor more well (e.g., microwells) where the wells (e.g., microwells) andthe surface do not have any polygonal angles (e.g., 90 degree angles).

In some embodiments, wells (e.g., microwells) have a cross-sectionalshape approximating a sine wave. In such embodiments, the bottom of thewell is rounded (e.g., hemispherically round), the side walls increasein diameter from the bottom of the well to the top and the boundarybetween wells is rounded. As such the top of the wells does notterminate at a right angle. In some embodiments, a well has a diameter Dat the half-way point (also termed D_(half-way)) between the bottom andtop, a diameter D_(top) at the top of the well and a height H frombottom to top of the well. In these embodiments, D_(top) is greater thanD. Both the relative and absolute dimensions of the wells may beselected for the desired culturing conditions. For spheroid growth, thediameter D is preferably one to three times the desired diameter of the3D cellular aggregate to be cultured in the well. The height H is 0.7 to1.3 times D. The diameter D_(top) is 1.5 to 2.5 times D. D is preferably100 micrometers (μm) to about 2000 micrometers (e.g., 100, 150, 200,250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1200, 1400,1600, 1800, or 2000 micrometers, including ranges between any two of theforegoing values (e.g., 200-1000 μm, 200-750 μm, 300-750 μm, 400-600 μm,etc.)). However, alternative relative or absolute dimensions may beemployed. For example, D may be from 1 to 10 times (e.g., 2, 3, 4, 5, 6,7, 8, 9) the desired diameter of the cellular aggregate or any value orrange therein between (e.g., 1, 1 to 1.5, 1 to 2, 2, 1 to 2.5, 1 to 3, 2to 3, 1 to 5, 3 to 5, 2 to 7, etc.). D may be from 100 μm to 10,000 μmor any value (e.g., 100, 200, 500, 1000, 2000, 5000) or range thereinbetween (e.g., 100-2000, 200-1000, 300-700, 400-600, 500, etc.). H maybe from 0.5 to 10 times D (e.g., 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2,2.5, 3, 4, 5, 6, 7, 8, 9, 10 or any values or ranges therein between).D_(top) may be from 1.1 to 5 times D (e.g., 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5 or any values or ranges therein between).

The barrier between contiguous wells (e.g., microwells) may have aninverse identical shape to the neighboring well, may have a larger orsmaller diameter D_(B) or may otherwise differ in shape (e.g., the shapeof the well bottom may differ from the shape of the well/barrier top,see e.g., FIG. 2A and FIG. 2B). To maximize the number of wells in agiven surface, D_(B) is preferably less than D. D_(B) may be from 1.1 to5 times larger or smaller than D (e.g., 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2, 3, 4, 5 or any values or ranges therein between).

In certain embodiments, cell culture apparatuses herein comprise aplurality of wells, each configured to cause cells cultured in the wellsto form spheroids of a specified diameter. The cell culture apparatuscan include a structure that defines a plurality of wells. In someembodiments, each of the plurality of wells defines a top aperture, awell-bottom, and a sidewall surface extending from the top aperture tothe well-bottom. The sidewall surface defines a pen tip area between thetop aperture and the well-bottom. A cell culture volume is defined bythe bottom surface, a portion of the sidewall surface, and the pen tiparea. In some embodiments, the pen tip area is defined by a diametricdimension in a range from 100 micrometers to 700 micrometers (e.g., 100μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, and any ranges therebetween) at a height in a range from 50 micrometers to 700 micrometers(e.g., 50 μm, 75 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700μm, and any ranges there between) from the well-bottom. The sidewallsurface or cell culture volume is dimensioned to control the size of aspheroid growing in each well. The pen tip area is a spheroid inducinggeometry.

Embodiments provide a number of features, including, for example: no airbubble entrapment during cell seeding or media exchange, high retentionof 3D cellular aggregates during media exchanges, ease of spheroidharvesting from large area surfaces, gas permeability, a media reservoirabove a plurality of wells, spheroid-confining wells, and/or the abilityto generate spheroids in large quantities and of uniform size.

In some embodiments, wells described herein comprise one or morecapillary structures (e.g., ridge, fissure, corner, acute angle,corrugation, pillar, etc.) that extend from the top opening inside thewell, and which may extend from the top opening to the well-bottom orfrom the top opening to the bottom of mouth, that provides a route forair to escape upon an influx of liquid into the well. Suitable wellgeometries within the scope of the embodiments described herein include:(a) wells having a square cross-section top opening, a rounded (e.g.,concave) well-bottom, and side walls that transition from a squarecross-section at the top of the well to a circular cross-section at thewell-bottom; (b) wells having a one or more protruding ridgelinesextending from the top opening (e.g., circular cross-section topopening) to the well-bottom (e.g., rounded (e.g., concave) well-bottom)(See, e.g., FIG. 1B); (c) wells having one or more fissures extendingfrom the top opening (e.g., circular cross-section top opening) to thewell-bottom (e.g., rounded (e.g., concave) well-bottom) (See, e.g., FIG.2B); (d) wells having an upper portion defined by first and secondsidewalls that fail to completely enclose the well, and a lower portionof the well with a rounded bottom and wherein the sidewalls completelycircumscribe the well (See, e.g., FIG. 27 or FIG. 28); (e) wells inwhich one or more sidewalls have a convex cross-section thereby creatingan acute angle between two sidewalls which may be formed by pillars(See, e.g., FIG. 5D). In some embodiments, wells comprise variationsand/or combinations of the above geometries.

In embodiments, provided herein are methods for making cell culturedevices comprising the wells described herein.

In embodiments, provided herein are methods for using the cell culturedevices comprising the wells described herein in, for example, spheroidcell culture or in cellular assays.

In some embodiments, provided herein are cell culture devices comprisinga frame having a well disposed therein, the well comprising: (a) a topopening; (b) a well-bottom having a rounded cross-sectional geometry;(c) a sidewall or sidewalls extending from the well-bottom to the topopening; (d) optionally a mouth; and (e) optionally a capillarystructure that facilitates the introduction of liquid into the well andthe escape of air from the well without the formation of persistent airpockets beneath the surface of the liquid.

In some embodiments, the well-bottom has a circular cross-sectionalgeometry, wherein the top opening has a polygonal cross-sectionalgeometry, and wherein the sidewalls transition from circular topolygonal cross-sectional geometry, thereby creating corners betweensidewalls that serve as the capillary structures that facilitates theintroduction of liquid into the well and the escape of air from thewell. In some embodiments, the transition of the sidewallcross-sectional geometry does not present obstructions to the flow offluid into or out of the well. In some embodiments, the top opening hasa square or hexagonal cross-sectional geometry.

In some embodiments, the structural feature that facilitates theintroduction of liquid into the well and the escape of air from the wellis a ridge protruding from the sidewall or sidewalls and extending fromwell-bottom to the top opening. In some embodiments, wells comprise 1-20ridges (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20) protruding from the sidewall or sidewalls and extending fromwell-bottom to the top opening, or substantially so. In someembodiments, the well-bottom has a circular cross-sectional geometry andthe top opening has a circular or polygonal cross-sectional geometry. Insome embodiments, the ridges are symmetrically spaced around theperimeter of the well. In some embodiments, the ridges areasymmetrically spaced around the perimeter of the well. In someembodiments, the ridges do not span the entire distance from top openingto well-bottom.

In some embodiments, the structural feature that facilitates theintroduction of liquid into the well and the escape of air from the wellis a fissure within the sidewall or sidewalls and extending fromwell-bottom to the top opening. In some embodiments, wells comprise 1-20fissures (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20) within the sidewall or sidewalls and extending fromwell-bottom to the top opening. In some embodiments, the well-bottom hasa circular cross-sectional geometry and the top opening has a circularor polygonal cross-sectional geometry. In some embodiments, the fissuresare symmetrically spaced around the perimeter of the well. In someembodiments, the fissures are asymmetrically spaced around the perimeterof the well. In some embodiments, the fissures do not span the entiredistance from top opening to well-bottom.

In some embodiments, the well is defined by 3 or more adjacent pillars(e.g., 3, 4, 5, 6, 7, 8, 9, 10), a portion of the side of each pillarforming the sidewalls of the well; and wherein a confined space betweenadjacent pillars forms the structural feature that facilitates theintroduction of liquid into the well and the escape of air from thewell. In some embodiments, the well-bottom has a circularcross-sectional geometry and extends below the pillars.

In some embodiments, provided herein are cell culture devices comprisinga frame comprising a plurality of wells disposed therein; wherein thewells are arranged in at least one row; wherein the row is defined bytwo corrugated sidewalls aligned to such that the gap between thesidewalls widens and narrows with each corrugation; wherein an upperportion of each well defined by a widened gap between two narrowed gapsof the sidewalls, such that the upper portion of adjacent wells are influid communication; and wherein a lower portion of each well extendsbelow the corrugated sidewalls and forms a circular well-bottom. In someembodiments, devices comprise multiple rows of wells.

In some embodiments, the sidewall, sidewalls, and/or well-bottom is gaspermeable and liquid impermeable. That is, in some embodiments, thesubstrate from which the wells are formed is gas permeable and liquidimpermeable.

In some embodiments, the sidewall or sidewalls is opaque and thewell-bottom is transparent.

In some embodiments, the well-bottom comprises a concave arcuatesurface.

In some embodiments, the sidewall, sidewalls, and/or well-bottomcomprises a low-adhesion or no-adhesion material and/or is coated with alow-adhesion or no-adhesion material.

In some embodiments, a cell culture device comprises from 8 to about10,000 wells (8, 16, 24, 32, 48, 64, 96, 128, 256, 384, 500, 600, 700,800, 1000, 1536, 2000, 2400, 3200, 4000, 10000, or any ranges therein).

In some embodiments, surfaces with microwell patterns are incorporatedinto a wide range of cell culture products. In some embodiments, thebottoms of wells (e.g., macrowells) in, for example, 12-, 24-, and6-well plates are patterned with microwell surfaces. In someembodiments, microwell surfaces are incorporated into large surface areacell culture vessels, such as T25, T75, T125, T175 and T250 flasks aswell as CellSTACK and HYPERStack lines of products. In some embodiments,culture of cells in large surface area vessels having a number of saidmicrowells yields large quantities of 3D cellular aggregates applicablein cell therapy applications, clonogenic culture, stem cell niches, orniche cells co-culture.

In some embodiments, cell culture devices herein comprise a bottom platedefining a major surface, one or more sidewalls extending from thebottom plate defining a reservoir, and a plurality of wells formed inthe major surface. Each well defines an upper aperture co-planar withthe major surface and open to the reservoir, and a well-bottom nadirpositioned below the major surface. In contrast to conventional wellplates, the plates described herein define a reservoir above the surfaceof the wells, which allows for increased volumes of cell culture mediato be used and thus provides for less frequent media exchange. See, forexample, FIG. 28.

In various embodiments, cell culture apparatuses having one or more cellculture compartments are described. In some embodiments, cell culturecompartments are stacked. In some embodiments, each cell culturecompartment includes a substrate defining a structured surface defininga plurality of gas permeable wells. In some embodiments, the wells arein gaseous communication with an exterior of the apparatus, eitherdirectly, via gas permeable materials, or via a vent or tracheal space.In some embodiments, cell culture apparatuses having, at least in part,a substrate having an array of microwells which wells are made from gaspermeable materials are described. Accordingly, in some embodiments, theapparatuses are used to culture cells within the wells while having aheight of cell culture media above the cultured cells that would be toohigh in existing cell culture apparatuses for efficient metabolic gasexchange. Because the cells are cultured in gas permeable wells that arein gaseous communication with the exterior of the apparatus, gasexchange occurs through the wells to overcome deficiency in gas exchangethrough the cell culture media due to the height of media above thecells.

In certain embodiments, a cell culture apparatus includes one or morecell culture compartments. In some embodiments, each cell culturecompartment has an interior and includes a substrate having a firstmajor surface and an opposing second major surface; the first majorsurface defines a structured surface within the interior of thecompartment. In some embodiments, the structured surface defines aplurality of gas permeable wells. In some embodiments, wells are ingaseous communication with an exterior of the apparatus.

In some embodiments, provided herein are methods of culturing spheroids,comprising: charging a cell culture device described herein with culturemedia; and adding spheroid forming cells to the culture media. In someembodiments, methods further comprise replacing/exchanging media (e.g.,daily, continuously, etc.).

In some embodiments, provided herein is the use of a cell culture devicedescribed herein for the culturing of spheroids.

Additional features and advantages of the subject matter of the presentdisclosure will be set forth in the detailed description which follows,and in part will be readily apparent to those skilled in the art fromthat description or recognized by practicing the subject matter of thepresent disclosure as described herein, including the detaileddescription which follows, the claims, as well as the appended drawings.

It is to be understood that both the foregoing general description andthe following detailed description present embodiments of the subjectmatter of the present disclosure, and are intended to provide anoverview or framework for understanding the nature and character of thesubject matter of the present disclosure as it is claimed. Theaccompanying drawings are included to provide a further understanding ofthe subject matter of the present disclosure, and are incorporated intoand constitute a part of this specification. The drawings illustratevarious embodiments of the subject matter of the present disclosure andtogether with the description serve to explain the principles andoperations of the subject matter of the present disclosure.Additionally, the drawings and descriptions are meant to be merelyillustrative, and are not intended to limit the scope of the claims inany manner.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B are schematic drawings of an exemplary embodiment ofan array of wells 100. FIG. 1A is an illustration in cross-section. FIG.1B is a top-down drawing of the exemplary embodiment of an array ofwells, taken at line B-B of FIG. 1A.

FIG. 2A and FIG. 2B are schematic drawings of another exemplaryembodiment of an array of wells 100. FIG. 2A is an illustration incross-section. FIG. 2B is a top-down drawing of the exemplary embodimentof an array of wells, taken at line B-B of FIG. 2A.

FIGS. 3A, 3B, 3C and 3D are schematic drawings of another exemplaryembodiment of an array of wells 100. FIG. 3A is an illustration incross-section. FIG. 3B is a top-down drawing of the exemplary embodimentof an array of wells, taken at line B-B of FIG. 3A. FIG. 3C is a drawingof an array of wells having a sinusoidal or parabolic shape. FIG. 3D isa side view of an array of wells containing spheroids, in an embodiment.

FIGS. 4A, 4B, 4C and 4D are schematic drawings showing another exemplaryembodiment of an array of spheroid-containing cell culture compartments,or wells, a corrugated embodiment. FIG. 4A and FIG. 4C are a top-downviews of a corrugated embodiment of a substrate having an array ofwells, and FIG. 4B and FIG. 4D are partial cut-away views of the sameembodiment.

FIGS. 5A, 5B, 5C, 5D and 5E show schematic drawings of an additionalexemplary embodiment in which a series of pillars define a well. FIGS.5A and 5B are top-down views and FIGS. 5C, 5D and 5E are partialcut-away perspective views of exemplary embodiments.

FIGS. 6A, 6B, 6C, 6D and 6E show exemplary, non-limiting examples ofcross-sectional geometrics for ridges protruding from sidewalls.

FIGS. 7A, 7B, 7C, 7D and 7E show exemplary, non-limiting examples ofcross-sectional geometrics for fissures within sidewalls.

FIG. 8 shows a culture flask having a bottom surface micropatterned withan array of microwells.

FIG. 9 shows a magnified view of the substrate, micropatterned with anarray of microwells, forming the bottom surface of the flask shown inFIG. 9.

FIG. 10A shows HT29 cellular spheroids inside the microwells of amicropatterned T25 spheroid forming flask, as shown in FIG. 9. FIG. 10Bshows harvested spheroids from a micropatterned T25 spheroid formingflask.

FIG. 11A and FIG. 11B show micrographs of spheroids or 3D aggregatesformed on a NUNCLON SPHERA™ low binding surface, available fromNunc/ThermoFisher. FIG. 11A shows human ESC cells and FIG. 11B showsmouse ESC cells.

FIG. 12 is an illustration of a method of making a substrate having amultiwell array, according to embodiments.

FIG. 13 shows a graph demonstrating viable cell counts measured aftergrowing cells in 6 well plates having substrates having an array ofmicrowells (as described in Example 1), inmicrowells with differentbottom thickness.

FIG. 14A and FIG. 14B show graphs comparing viable cell count (FIG. 14A)and cell productivity (FIG. 14B) for substrates having arrays ofmicrowells versus flat surfaces.

FIG. 15 shows a graph depicting total protein titer excreted from MH677cells cultured on substrates having arrays of microwells versus flatsurface.

FIG. 16 is an image of an embodiment of a structured surface.

FIG. 17 is a photograph of cells grown in wells of an embodiment of astructured surface.

FIG. 18 is a side view illustrating an embodiment of a cell cultureapparatus including a porous membrane support.

FIG. 19 is a side view illustrating an additional embodiment of a cellculture apparatus including a porous membrane support.

FIG. 20 is a side view illustrating an additional embodiment of a cellculture apparatus including a porous membrane support illustratingco-culture of cells.

FIG. 21 is a schematic perspective view of an embodiment of a cellculture apparatus.

FIG. 22 is a schematic cross-sectional view of an embodiment of a cellculture apparatus.

FIG. 23 is a schematic cross-sectional view of an embodiment of a cellculture apparatus.

FIG. 24A is a schematic bottom view of an embodiment of a tray that canbe used to form a part of an apparatus as depicted in any of FIGS. 21,22 and 23.

FIG. 24B is a schematic perspective view of an embodiment of the trayshown in FIG. 24A.

FIG. 25 is a schematic side view of an embodiment of a cell cultureapparatus.

FIG. 26 is a perspective view of an embodiment of a cell cultureapparatus having wells.

FIG. 27 is a schematic cross-sectional view of an embodiment of aplurality of wells.

FIG. 28 is a schematic cross-sectional view of an embodiment of aplurality of wells.

FIG. 29 is a magnified schematic cross-sectional view of an embodimentof a plurality of wells.

FIG. 30 is a schematic perspective view of an embodiment of a cellculture apparatus having a plate and wells.

FIG. 31 is a schematic perspective view of an embodiment of a cellculture apparatus having a plate and wells.

FIG. 32 is a schematic perspective view of an embodiment of a cellculture apparatus and an insert including a mesh.

FIG. 33 is schematic a perspective view of an embodiment of an apparatushaving an inlet and an outlet.

FIG. 34A and FIG. 34B show graphs depicting Protein production per cm²in 96-well spheroid micro-well plates (FIG. 34A) in CHO 5/9 alpha cells,and (FIG. 34B) in BHK-21 pc.DNA3-1HC cells.

DETAILED DESCRIPTION

Various embodiments of the disclosure will be described in detail withreference to drawings. Reference to various embodiments does not limitthe scope of the invention. Additionally, any examples set forth in thisspecification are not limiting and merely set forth some of the manypossible embodiments of the claimed invention. Like numbers used in thefigures refer to like components, steps and the like. However, it willbe understood that the use of a number to refer to a component in agiven figure is not intended to limit the component in another figurelabelled with the same number. In addition, the use of different numbersto refer to components is not intended to indicate that the differentnumbered components cannot be the same or similar to other numberedcomponents.

All scientific and technical terms used herein have meanings commonlyused in the art unless otherwise specified. The definitions providedherein are to facilitate understanding of certain terms used frequentlyherein and are not meant to limit the scope of the present disclosure.

In certain embodiments, the apparatuses herein and the methods of makingand using such apparatuses provide one or more advantageous features oraspects, including for example as discussed below. Features or aspectsrecited in any of the claims are generally applicable to all facets ofthe invention. Any recited single or multiple feature or aspect in anyone claim can be combined or permuted with any other recited feature oraspect in any other claim or claims.

“Include,” “includes,” or like terms means encompassing but not limitedto, that is, inclusive and not exclusive.

“About” modifying, for example, the quantity of an ingredient in acomposition, concentrations, volumes, process temperature, process time,yields, flow rates, pressures, viscosities, and like values, and rangesthereof, or a dimension of a component, and like values, and rangesthereof, employed in describing the embodiments of the disclosure,refers to variation in the numerical quantity that can occur, forexample: through typical measuring and handling procedures used forpreparing materials, compositions, composites, concentrates, componentparts, articles of manufacture, or use formulations; through inadvertenterror in these procedures; through differences in the manufacture,source, or purity of starting materials or ingredients used to carry outthe methods; and like considerations. The term “about” also encompassesamounts that differ due to aging of a composition or formulation with aparticular initial concentration or mixture, and amounts that differ dueto mixing or processing a composition or formulation with a particularinitial concentration or mixture.

“Optional” or “optionally” means that the subsequently described step,feature, condition, characteristic, or structure, occurs/is present ordoes not occur/is not present, while still being within the scopedescribed.

The words “preferred” and “preferably” refer to embodiments of thedisclosure that may afford certain benefits, under certaincircumstances. However, other embodiments may also be preferred, underthe same or other circumstances. Furthermore, the recitation of one ormore preferred embodiments does not imply that other embodiments are notuseful, and is not intended to exclude other embodiments from the scopeof the inventive technology.

The devices, the methods of making the devices, and the method of usingthe devices, described herein may include components or steps describedherein, plus other components or steps not described herein.

As used in this specification and the appended claims, the term “or” isgenerally employed in its sense including “and/or” unless the contentclearly dictates otherwise. The term “and/or” means one or all of thelisted elements or a combination of any two or more of the listedelements.

As used herein, “have”, “has”, “having”, “include”, “includes”,“including”, “comprise”, “comprises”, “comprising” or the like are usedin their open ended inclusive sense, and generally mean “include, butnot limited to”, “includes, but not limited to”, or “including, but notlimited to”.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, examples include from the one particular value and/or to theother particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it will be understoodthat the particular value forms another aspect. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint.

The indefinite article “a” or “an” and its corresponding definitearticle “the” as used herein means at least one, or one or more, unlessspecified otherwise.

Abbreviations, which are well known to one of ordinary skill in the art,may be used (e.g., “h” or “hrs” for hour or hours, “g” or “gm” forgram(s), “mL” for milliliters, and “rt” for room temperature, “nm” fornanometers, and like abbreviations).

Specific and preferred values disclosed for components, ingredients,additives, dimensions, conditions, and like aspects, and ranges thereof,are for illustration only; they do not exclude other defined values orother values within defined ranges, unless otherwise noted. Theapparatus and methods of the disclosure include any value or anycombination of the values, specific values, more specific values, andpreferred values described herein, including explicit or implicitintermediate values and ranges there between.

Also herein, the recitations of numerical ranges by endpoints includeall numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.80, 4, 5, etc.). Where a range of values is “greater than”,“less than”, etc. a particular value, that value is included within therange.

Any direction referred to herein, such as “top,” “bottom,” “left,”“right,” “upper,” “lower,” “above,” below,” and other directions andorientations are described herein for clarity in reference to thefigures and are not to be limiting of an actual device or system or useof the device or system. Many of the devices, articles or systemsdescribed herein may be used in a number of directions and orientations.Directional descriptors used herein with regard to cell cultureapparatuses often refer to directions when the apparatus is oriented forpurposes of culturing cells in the apparatus.

Unless otherwise expressly stated, it is in no way intended that anymethod set forth herein be construed as requiring that its steps beperformed in a specific order. Accordingly, where a method claim doesnot actually recite an order to be followed by its steps or it is nototherwise specifically stated in the claims or descriptions that thesteps are to be limited to a specific order, it is no way intended thatany particular order be inferred. Any recited single or multiple featureor aspect in any one claim can be combined or permuted with any otherrecited feature or aspect in any other claim or claims.

It is also noted that recitations herein refer to a component being“configured” or “adapted to” function in a particular way. In thisrespect, such a component is “configured” or “adapted to” embody aparticular property, or function in a particular manner, where suchrecitations are structural recitations as opposed to recitations ofintended use. More specifically, the references herein to the manner inwhich a component is “configured” or “adapted to” denotes an existingphysical condition of the component and, as such, is to be taken as adefinite recitation of the structural characteristics of the component.

While various features, elements or steps of particular embodiments maybe disclosed using the transitional phrase “comprising,” it is to beunderstood that alternative embodiments, including those that may bedescribed using the transitional phrases “consisting” or “consistingessentially of” are implied. Thus, for example, implied alternativeembodiments to a cell culture apparatus comprising a structure defininga plurality of wells include embodiments where cell culture apparatusconsists of a structure defining a plurality of wells and embodimentswhere a cell culture apparatus consists essentially of a structuredefining a plurality of wells.

In various embodiments, the disclosure describes devices, such as cellculture apparatuses, including a substrate defining a well (e.g., amicrowell). The well comprises a sidewall (or sidewalls), well-bottom(or nadir), and an open top (e.g., upper aperture). In embodiments, thewell is configured to contain an aqueous liquid composition, forexample, a composition employed in cell culture or cell assays. Forexample, an aqueous liquid composition may include a cell culturemedium, buffers or other solutions or mixtures employed in cell assays.

The embodiments described herein find use, for example, with any devicethat comprises small (e.g., microscale) wells or other vessels orchambers that are configured to contain liquid. In particularembodiments, wells of devices described herein find use in the cultureof cells. More particularly, the devices and microwells therein find usein the 3D cell culture of cell aggregates or spheroids.

There are some different geometries that have been used for the cultureof cells as aggregates. In some embodiments, cell aggregates areclusters of cells, embryoid bodies, or spheroids. A common geometry toform cell aggregates are hemispheres found on rounded well-bottommicroplates. In some embodiments, a non-adhesive surface is used toprevent the cells from attaching to the surface. A non-adhesive materialmay be applied after a well or chamber (e.g., in a microplate) ismanufactured, or the well or chamber material may have inherentnon-attachment characteristics.

FIG. 1A and FIG. 1B are is a schematic drawings of an exemplaryembodiment of an array of wells 100, showing individual wells 115. Inthe embodiment illustrated in FIGS. 1A and 1B, well 115 has a mouth 110.Mouth 110 is a region at the top part of the well, adjacent the topopening 111 of the well 115, which provides a more open area, before thewell constricts to form a well-bottom where cells settle to formspheroids. In embodiments, mouth 110 can be conical (wider at the top ofthe mouth than at the bottom of the mouth) and annular in shape (asshown in FIG. 1A and FIG. 2A, where the well is round). In additionalembodiments, as shown in, for example, FIG. 3A, where the well has around opening, but is parabolic in shape, mouth 110 may be parabolic oras shown in FIG. 27 and FIG. 28, mouth may extend into each well 115. Inembodiments, mouth is absent. The presence of a mouth structure canprovide two functions. First, the mouth widens the opening of the well,and allows liquid introduced into the opening of the well to flow downto the bottom of the well. This promotes the aggregation of cells at thebottom of the well and promotes the formation of spheroids in culture.In addition, mouth creates a transition between the annular internalsurface of mouth to the internal surface of the well, thereby providinga geometric feature that may prevent the entrapment of air in the well.The presence of a 90 degree angle between the top of a well and thesidewall of a well may provide a location for formation of an airbubble. The mouth provides a transition between the top of a well and asidewall that is not a 90 degree angle, thereby reducing the formationof air bubbles in a well having a mouth structure.

Well dimensions for use in aggregate cell culture techniques may be onthe order of micrometers to millimeters (e.g., 100 μm to 50 mm).Well-containing devices for cell culture are sold by many differentmanufacturers (e.g., Corning, Nunc, Greiner, etc). “Microwells” arewells generally having dimensions on the order of micrometers (e.g., ≦1mm, ≦500 μm, ≦400 μm, ≦200 μm) or a few millimeters (e.g., ≦10 mm, ≦5mm, ≦3 mm, etc.), and are also used to grow cells as aggregates. In someembodiments, microwells provide confinements for 3D cell culture. In anysuitable embodiments herein, the term “well” encompasses the use ofmicrowells, except where described or indicated otherwise by context(e.g., where a well is described as having a well-bottom comprising aplurality of microwells). Wells intended to be outside the microwelldimensions may be referred to as “macrowells” or simply as “wells.” Insome embodiments, well height or well-depth (e.g., from top aperture towell-bottom) is equal to 100% or greater the well-diameter at topaperture (e.g., 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%,190%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, or anyranges there between. In some embodiments, well-depth (e.g., from topaperture to well-bottom) is equal to 100% or greater the well-diameterat the midpoint between the top aperture and well-bottom (e.g., 100%,110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 225%, 250%,275%, 300%, 325%, 350%, 375%, 400%, or any ranges there between.

One of the most commonly used microwell products is the commerciallyavailable “Aggrewell” plate (sold by Stem Cell technologies), whichoffers a geometry that is an inverse pyramidal shape 400 or 800micrometers in diameter arrayed in the bottom of standard formatmicroplate wells. Another geometry for growing cells as aggregates isthe “Elplasia” microplate with “microspace cell culture” (Kuraray);these plates have square microwells 200 micrometers in diameter arrayedat the bottom of standard format microplate wells that allow cells toaggregate. Various parameters, dimensions, and methods of makingmicrowells for culturing cells as aggregates are understood in the field(U.S. Pub. No. 2004/0125266; U.S. Pub. No. 2012/0064627; U.S. Pub. No.2014/0227784; WO2008/106771; WO2014/165273; herein incorporated byreference in their entireties). U.S. Pat. No. 6,348,999 describes microrelief elements, and how they are constructed, without stating thepurpose of these constructs other than as a polymer lens array. U.S.Pat. Nos. 5,151,366, 5,272,084, and 6,306,646 describe vessels withvarious types of micro relief patterns to increase the surface area forcell attachment on a substrate, and the method of making the culturepatterns, but the patterns themselves would not be conducive to theformation of cell aggregates. Other devices, compositions, reagents andmethods have been described in the art, for example, U.S. Pub. No.2014/0322806; U.S. Pat. No. 8,906,685; Haycock. Methods Mol Biol. 2011;695:1-15.; U.S. Pub. No. 2014/0221225; WO 2014/165273; U.S. Pub. No.2009/0018033; herein incorporated by reference in their entireties.

Some commercially available well geometries are conducive to theformation of cell aggregates, but not necessarily conducive to“confinement”. When aggregated cells are not confined, they will usuallygrow as large as their surroundings will allow. Cell aggregates greaterthan 150 to 400 micrometers in diameter (depending on the cell type) mayform necrotic cores. Necrosis occurs, for example, because the cell massis so large that the diffusion of nutrients into the center of theaggregate and the metabolic waste out of the center of the aggregate islimited. In some embodiments, in order to create confinement, amicrowell geometry is used that is very similar (e.g., within 50%, 40%,30%, 20%, 15%, 10%, 5%, 2%, 1%, or suitable ranges therein) to the sizeof the maximum desired cell aggregate in diameter, but at least 1.5 to 2times the diameter in depth (e.g., 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1,2.2, 2.3, 2.4, 2.5 2.6, 2.6, 2.8, 3.0, 3.5, 4.0, or suitable rangestherein). In some embodiments, confinement well geometry also allows forexchange of liquid culture medium through perfusion or manual pipettingwithout lifting the cell spheroids out of the confinement wells.

Existing devices (e.g., microwell format microplates or other vesselswith microwells) exhibit a design flaw that negatively impacts the useof such devices for the formation of 3D cell aggregate structures.Although handling of microwell format microplates is fairlystraightforward, failure of air to be displaced from microwells uponintroduction of liquid (e.g., media) often poses problems. Airentrapment is a common problem in microplate wells with geometry aslarge as 384-well formats, particularly if the wells are circular. Thesurface tension of the aqueous liquid is strong so drops remainspherical. A spherical drop is capable of blocking a circular hole(e.g., well cross-sectional geometry) of similar size. The presence ofair within the well will negatively impact and/or inhibit the culture ofcells within that well.

Growing spheroids in cell culture vessels at high density on substrateswith arrays of spheroid formation wells having non-adherent surfacesrequires culture surfaces that balance many variables. A design that canbalance, for example, maximum achievable spheroid density, ability tomaintain spheroids in location during fluid exchange activities whilebeing able to remove them when desired, and avoiding the entrapment ofair in the spheroid formation wells when the vessel is filled is highlydesirable to avoid difficulty in working with such vessels. Embodimentsherein address the air entrapment problem of traditional wells byproviding well geometries that will help displace air in microwells andpermit the entrance of liquid into the microwells, while maintainingconfinement dimensions. For example, square top wells with roundedwell-bottom geometry are significantly less likely to entrap air whenliquid is added, since the air is able to rise up the corners of thewell around the aqueous droplet. In some embodiments, the inclusion ofvarious structures (e.g., pillars, corrugations, corners, ridges,fissures, etc.) extending from the opening of the well to thewell-bottom provides pathways for air escape upon introduction of liquidinto the wells. In some embodiments, in addition to the featuresdescribed herein, also comprise geometries, materials, etc. that aredescribed in the art and/or understood in the field.

To avoid the issue of air entrapment in high density spheroid growthsubstrates, one design feature that has commonly been utilized is theavoidance of sharp corners or step changes in the substrate geometry,particularly ones that run orthogonal to the flow path of liquid acrossthe surface. For example, the Aggrewell plate has walls that drop off atnear 90 degree angles. This promotes liquid breaking from the surface asthe vessel is filled leaving wells that are filled with air.

Provided herein are surface geometries that address the problem of airentrapment during liquid introduction, maintaining well features thatpromote growth and maintenance of high densities of discrete spheroids(e.g., rounded well-bottoms).

In some embodiments, well-shape transitioning is utilized to alleviateissues with air-escape upon introduction of liquid into the wells. Forexample, in some embodiments, a circular cross-section well-bottom (orbottom portion of the well) is utilized for spheroid formation. However,the circular cross-section can be particularly problematic for airescape without pocket formation. To alleviate this issue, wells areformed with a circular well-bottom cross-section and a non-circular(e.g., triangular, square, rectangular, pentagonal, hexagonal, etc.) topopening. In such embodiments, the sidewalls transition from thenon-circular (e.g., polygonal) top opening to the circular well-bottom.In some embodiments, the transition is a gradual one, so as to notintroduce any interfering, jagged, or horizontal-presenting sidewallfeatures that could result in the ‘hanging up’ of air bubbles escapingthe well upon introduction of liquid to the well. In some embodiments,the corners in the sidewalls created by the non-circular (e.g.,polygonal) shape of the transitioning walls and top opening providepathways for the entry of liquid and/or the escape of air.

In some embodiments, well geometries comprise capillary structures(including, for example, a mouth, ridge fissure, rounded or parabolictop opening, etc.) in the well walls to facilitate the escape of airupon introduction of liquid into the well. FIG. 1B is a top-down view,taken at line B-B of FIG. 1A, illustrating a ridge 170. As shown in FIG.1B, the ridge is a bump or a protuberance from the mouth 110 or thesidewall 113 of the well. In embodiments, the ridge extends the lengthof the microwell from the top opening 111 to the well bottom 116. Inadditional embodiments, the ridge extends from the top of the mouth 111to the bottom of the mouth 112. The sharp angles formed on either sideof the ridge 170 create a capillary force on the aqueous fluid toprovide for fluid entry to the microwell without air entrapment. FIG. 2Bis a top-down view of an array of wells 100 shown in cross section inFIG. 2A. FIG. 2B illustrates a fissure 270. As shown in FIG. 2B, thefissure is an indentation in the sidewall 113 of the well 115. The sharpangles formed on either side of the fissure 270 create a capillary forceto allow aqueous fluid flow into the microwell.

FIG. 3A, FIG. 3B, and FIG. 3C are schematic drawings of anotherexemplary embodiment of an array of wells 100. FIG. 3A is anillustration in cross-section. FIG. 3B is a top-down drawing of theexemplary embodiment of an array of wells, taken at line B-B of FIG. 3A.FIG. 3 A and FIG. 3B illustrate that each well 115 may have more thanone ridge 170 or fissure 270, and that ridges 170 or fissures 270 may bearranged in an array within the well 115. As shown in FIG. 3A and FIG.3B, in embodiments, a radial distribution of ridges and/or fissures isenvisioned. The number of capillary structures is not limited to one permicrowell. In some embodiments, greater numbers of capillaries increasethe rate of fluid entry into the microwell.

FIGS. 3A, 3B, 3C and 3D demonstrates inclusion of multiple (e.g., 2, 3,4, 5, 6, 7, 8, 9, 10, 12, 16, 20, 24, 28, 32, or any ranges therein)vertically-oriented capillary structures within a single well. Featuresmay be regularly-spaced (as depicted in FIGS. 3A, 3B, 3C and 3D),irregularly spaced, grouped/bunched, etc. In some embodiments, capillarystructures extend from the top opening of the well to the well bottom.When multiple capillary structures are present in a single well, themultiple features may be of different types (e.g., ridgelines and/orfissures) and may comprise different shapes (e.g., square, rounded,etc.)

FIG. 3C illustrates that the array of wells 100 may have a sinusoidal orparabolic shape. This shape creates a rounded top edge or well edgewhich, in embodiments, reduces the entrapment of air at a sharp corneror 90 degree angle at the top of a well. This sinusoidal or parabolicwell shape, or rounded top well edge, is also a capillary structure. Asshown in FIG. 3C, the well 115 has a top opening having a top diameterD_(top), a height from the bottom of the well 116 to the top of the wellH, and a diameter of the well at a height half-way between the top ofthe well and the bottom 116 of the well D_(half-way).

FIG. 3D is a schematic drawing of an array of wells, as described abovein FIGS. 1A, 1B, 2A, 2B, 3A, 3B, 3C and 3D, and in embodiments. FIG. 3Dillustrates a plurality of microwells 115 arranged in an array in asubstrate 1114. Also shown in FIG. 3D are a plurality of spheroids 500residing in the plurality of microwells 115.

FIG. 4A and FIG. 4B are schematic drawings showing another exemplaryembodiment of an array of spheroid-containing cell culture compartments,or wells. FIG. 4A is a top-down view of a corrugated embodiment of anarray of wells, and FIG. 4B is a partial cut-away view of the sameembodiment. In the embodiment shown in FIG. 4A and FIG. 4B, the wellsare not isolated, but allow for the flow of liquid between wells. Asshown in FIGS. 4A and 4B an exemplary embodiment is illustrated in whichcorrugated sidewalls 403 are aligned to produce microwells 401 in thegaps between the corrugated sidewalls 403. The corrugated sidewalls,shown in FIG. 4A and FIG. 4B are enclosed by a frame 402, have regionswhere they are far apart and then come closer together (e.g., with orwithout contact) in a periodicity that creates rows of microwells 401.In some embodiments, a microwell depression lies at the base of eachsegment that is defined by the walls being farther apart (e.g., to housea spheroid 500). FIG. 4C and FIG. 4D are additional schematic drawingsof the corrugated microwell array embodiment. FIG. 4C is a top downdrawing, and FIG. 4D is perspective view of the embodiment. As shown inFIG. 4D, in embodiments, no frame is present. FIG. 4C and FIG. 4Dillustrate corrugated or wavy sidewalls 403 which form spaces havinggeometry suitable for inducing spheroid formation, 401. Also shown inFIG. 4C and FIG. 4D are spheroids 500 residing in the wells. When liquidis introduced into the embodiment shown in FIG. 4A and FIG. 4B,displaced air can move out of the local area through the areas where thewalls are closer together. Movement of fluid and air to and from thewider well areas is promoted by the narrow sections, to avoid airentrapment. These corrugations also promote the formation of spheroidsby providing constricted growth areas. That is corrugations are bothcapillary structures and spheroid inducing geometry.

FIG. 5A and FIG. 5B show top-down schematic drawings of exemplaryembodiments of an array of wells 100 in which a series of pillars definea well. FIGS. 5C, 5D and 5E are perspective drawings of embodiments ofan array of microwells showing pillars 501 arranged in arrays to formwells 515. In embodiments, the tops 510 of the pillars 501 may be flat(as shown in FIG. 5A). In this embodiment, convex walls created by thepillars 501 create very acute angles or discontinuities at the junctionbetween the pillar-formed sidewalls. FIG. 5B depicts the microwelldepressions surrounded by pillars to create a confinement geometrysuitable to induce the formation of spheroids 500 in culture. Airescapes and fluid enters through spaces 505 between the pillars 501. Thepillars 501 have tops 510. In embodiments, the pillar tops 510 may berounded, as shown in FIG. 5B, FIG. 5D and FIG. 5E, which would result inwells having the parabolic or sinusoidal shape shown in FIG. 3A. Pillarsalso promote the formation of spheroids by providing constricted growthareas. That is, pillars are both capillary structures and spheroidinducing geometry.

These structures, including, for example, ridges, fissures, bumps,divots, open ring structures at the top aperture or mouth structure,pillars, discontinuous walls, mouth structures, parabolic or sinusoidalwell shape, rounded well opening, or interruptions in the smoothinternal surface of a sidewall of a well, or a combination of any ofthese features, are capillary structures. Capillary structures alsoprovide routes of escape for any air that might become trapped followingaddition of liquid. In some embodiments, discontinuous walls, walls thatcontain ridges or fissures or other features that interrupt thesmoothness of the sidewall of a well, are used to avoid air entrapmentby providing venting locations within the wells during the fill.

In some embodiments, capillary structures extend along the verticallength of walls of the wells. In some embodiments, capillary structuresextend up to or above the top opening of a well. In other embodiments,capillary structures extend near the top opening of a well (e.g., <0.1,0.1 μm, 0.2 μm, 0.5 μm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 10 μm, or 20 μmfrom the top opening (or ranges there between)). In some embodiments,capillary structures extend to the well-bottom. In other embodiments,capillary structures extend near the well-bottom (e.g., <0.1, 0.1 μm,0.2 μm, 0.5 μm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 10 μm, or 20 μm from thewell-bottom (or ranges there between)).

In some embodiments, capillary features provide the benefit of providingpathways for liquid entering the well to travel without entrapping airwithin the well. In some embodiments, capillary structures providepathways for air to exit the well in response to the introduction ofliquid entering the well. The technology is not limited to anyparticular mechanism of action for prevention of air entrapment, and anunderstanding of the mechanism is not necessary to practice the presentinvention.

In the event that rapid vessel fill or another fluid-introduction eventleads to air entrapment, despite well geometry configured to preventsuch entrapment, capillary features allow transfer of liquid under theentrapped air, thereby releasing the air from the well. For example, inthe corrugated embodiment shown in FIGS. 4A, 4B, 4C and 4D, liquid andair can flow through the open well structure from one well area to thenext well area through the narrowed sections of the array. Or, in thepillar embodiment shown in FIGS. 5A, 5B, 5C, 5D and 5E, liquid orentrapped air can leave a well through the spaces between pillars. Insome embodiments, the downward force of liquid through the capillarystructures serves to separate the air from the well wall, surroundingthe air with liquid, so the air pocket rises out of the well as abubble.

A variety of different vertically-oriented structures find use ascapillary features. For example, in certain embodiments, features areraised ridges (e.g., as depicted in FIG. 1A and FIG. 1B), or sunkengrooves or fissures (e.g., as depicted in FIG. 2A and FIG. 2B).Ridgelines and fissures that find use in embodiments herein are notlimited to the physical geometries depicted in the figures. In someembodiments, features extend vertically along the sidewall of the well,without traversing horizontally. In most situations, horizontalstructural features promote air entrapment within the wells in a similarfashion to the steep angles used in existing well geometries. Suitablecross-sectional geometries for ridges are depicted in FIGS. 6A, 6B, 6C,6D and 6E and include: rounded (FIG. 6A), angular (FIG. 6B), needle(FIG. 6C), hemi-hexagonal (FIG. 6D and FIG. 6E), etc. Suitablecross-sectional geometries for fissures are depicted in FIGS. 7A, 7B,7C, 7D and 7E and include: rounded (FIG. 7A), angular (FIG. 7B), needle(FIG. 7C), hemi-hexagonal (FIG. 7D and FIG. 7E), etc. Ridges and/orfissures may be of any suitable cross-sectional dimensions (e.g., havingwidths, lengths, etc. of 0.1-20 micrometers (e.g., 0.1, 0.2, 0.5, 1, 2,5, 10, 15, 20, or any suitable ranges there between). Engineering andmicrofluidic principles may be used in combination with embodimentsherein to optimize ridge and/or fissure shape and dimensions tofacilitate the introduction of liquid and the exit of air without theformation of air pockets beneath the liquid surface and/or to facilitatethe removal trapped air pockets.

In some embodiments, transfer of liquid into and air out of a well ismediated by discontinuous sidewall geometry. Discontinuous wellgeometries take the form of discontinuities in sidewalls of the wells.Examples of discontinuous sidewall geometries are as depicted in the“wave wall” or “corrugated” geometry of FIGS. 4A, 4B, 4C and 4D and the“pin wall” or “pillar wall” geometry of FIGS. 5A, 5B, 5C, 5D and 5E.These geometries are only exemplary; other sidewall orientations thatintroduce a gap or other discontinuity into a portion (e.g., upperportion) of the sidewall(s) may find use in embodiments herein. In thesegeometries, the interruption(s) in the wall allow air, with its lowviscosity, to rapidly move out of the wells as the fluid enters thevessel. In some embodiments, discontinuous geometries maintain theavoidance of sharp angle changes in the substrate wall features (e.g.,avoidance of features that create horizontal obstructions). In someembodiments, discontinuous sidewall geometries are used in conjunctionwith well-shape transitioning and/or capillary wall structure to aidbubble release and/or air exit upon liquid introduction.

In some embodiments, one or more wells have a concave surface, such as ahemi-spherical surface or a conical surface having a rounded bottom, andlike surface geometries or a combination thereof. The well and wellbottom can ultimately terminate, end, or bottom-out in a rounded orcurved surface, such as a dimple or a well, and like concavefrusto-conical relief surfaces, or combinations thereof. Other shapesand construction of spheroid-conducive wells are described incommonly-assigned U.S. patent application Ser. No. 14/087,906, whichapplication is hereby incorporated herein by reference in its entiretyto the extent that it does not conflict with the present disclosure. Inembodiments, well bottoms are flat or come to a point. Well bottoms mayhave any other suitable shape or dimension. For example, in embodiments,well bottoms have rounded or curved surfaces, or well bottoms may havestructures such as a dimple, a pit, and like, concave frusto-conicalrelief surfaces, dimples, or pen-tip areas or combinations thereof whichpromote the formation of spheroids by providing constricted growthareas. That is, rounded or curved or dimpled well bottoms, or pen tipareas or corrugations or pillars are spheroid inducing geometry.

Exemplary well geometry and size is depicted, for example, in FIG. 1A,FIG. 1B, FIG. 2A and FIG. 2B. In some embodiments, the wells 100described herein have well-bottom diameters 130/230 ranging from about100 micrometers to about 2000 micrometers, e.g., 100, 150, 200, 250,300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600,1800, or 2000 micrometers, including ranges between any two of theforegoing values (e.g., 200-1000 μm, 200-750 μm, 300-750 μm, 400-600 μm,etc). Such diametric dimensions control the size of a spheroid growntherein such that cells at the interior of the spheroid are maintainedin a healthy state. That is, these dimensions promote the formation ofspheroids by providing constricted growth areas. That is, thesedimensions are spheroid inducing geometry.

In some embodiments, the wells 100/200 described herein have top-openingcross-sectional dimensions 120/220 (e.g., diameter or width(s)) in arange from about 100 micrometers to about 2000 micrometers, e.g., 100,150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1200,1400, 1600, 1800, or 2000 micrometers, including ranges between any twoof the foregoing values. In some embodiments, the wells 100 have a depth160/260 from top opening to well-bottom in a range from about 500micrometers to about 1500 micrometers, e.g., 100, 200, 300, 400, 500,600, 700, 800, 900, or 1000 micrometers, including ranges between anytwo of the foregoing values. In some embodiments, the wells 100/200 havean upper portion with a depth 140/240 in a range from about 50micrometers to about 500 micrometers, e.g., 50, 60, 70, 80, 90, 100,200, 300, 400, or 500 micrometers, including ranges between any two ofthe foregoing values. In some embodiments, the wells 100 have a lowerportion with a depth 150/250 in a range from about 100 micrometers toabout 1400 micrometers, e.g., 100, 150, 200, 250, 300, 350, 400, 450,500, 600, 700, 800, 900, 1000, 1200, or 1400 micrometers, includingranges between any two of the foregoing values. Of course, othersuitable dimensions may also be employed.

In some embodiments, in addition to transition well structures andcharacteristics, as well as one or more design elements and/or physicalfeatures configured to allow escape of air upon introduction of liquidto a well, the devices and wells (e.g., microwells) described herein maycomprise additional features to provide specialized functionality, forexample, related to the 3D culture of cells within the microwells. Thefollowing paragraphs address such features that may find use incombination with the embodiments discussed above.

In some embodiments, all or a portion of the sidewalls and orwell-bottom of a well is gas permeable. In some embodiments, gaspermeability allows for transfer of oxygen and other gases into the wellto be dissolved into the liquid or media contained within the well. Thepermeable sidewalls, well-bottoms, or portions thereof do not allow forthe formation of air pockets or bubbles in the well liquid.

In some embodiments, cell culture apparatuses are provided having astructured surface defining a plurality of gas permeable wells. In someembodiments, the wells can comprise an exterior surface that defines anexternal surface of the apparatus. In some embodiments, the wells cancomprise an exterior or non-culture surface that is in communicationwith an exterior of the apparatus. In some embodiments, provided hereinare, among other things, cell culture apparatuses having a plurality ofstacked cell culture compartments, each having a structured surfacedefining a plurality of gas permeable wells. In some embodiments, thewells in various embodiments are in gaseous communication with anexterior of the apparatus, such as indirectly through a vent or througha tracheal space, or directly through an exterior wall.

In some embodiments, wells are configured such that cells cultured inthe wells form spheroids. For example, in some embodiments, the wellsare non-adherent to cells to cause the cells in the wells to associatewith each other (e.g., to form spheroids). The spheroids expand to sizelimits imposed by the geometry of the wells. In some embodiments, thewells are coated with an ultra-low binding material to make the wellsnon-adherent to cells.

The formation of three-dimensional (3D) cell agglomerates such asspheroids, as opposed to two-dimensional cell culture in which the cellsform a monolayer on a surface, increases the density of cells grown in acell culture apparatus, which can in turn increase nutrient demands ofthe cells cultured in the apparatus. Because metabolic gas exchange canoccur through the gas permeable wells in which the cells are cultured,media volume in the cell culture apparatus can be greater than ispossible with apparatuses in which metabolic gas exchange is essentiallylimited to diffusion through the cell culture medium. Accordingly, alarger cell culture medium height, and thus volume, can be used withcell culture apparatuses described herein.

In some embodiments, cells are cultured in wells of apparatusesdescribed herein where the cell culture medium is at a height of 2 mm ormore above the cells. In some embodiments, cell culture medium is at aheight of 5 mm or more above the cells. A maximum cell culture mediumheight of 2 mm to 5 mm is generally considered an upper limit of mediumheight when metabolic gas exchange is essentially limited to through themedium, such as when the substrate or surface on which, or near which,the cells are cultured is impermeable or relatively impermeable (e.g.,as compared to the cell culture medium) to metabolic gases.

For purposes of efficient metabolic gas exchange, in some embodiments,cells are maintained in culture in wells of apparatuses described hereinwhen the cell culture medium in the apparatus is at any height above thecells, such as 2 mm or more above the cells, 5 mm or more above thecells or 10 mm or more above the cells. However, one of skill in the artwill understand that as the height of cell culture medium in anapparatus increases above the cells, hydrostatic pressure exerted on thecells increases. Accordingly, there may be practical limitations to theheight of cell culture medium above the cells. In some embodiments, theheight of cell culture medium above cells cultured in wells of articlesdescribed herein is in a range from 5 mm to 20 mm, such as 5 mm to 15mm, 6 mm to 15 mm, 5 mm to 10 mm or 6 mm to 10 mm, such as 5, 6, 7, 8,9, 10, 15 or 20 mm, including ranges between any two of the foregoing.

In certain embodiments, a cell culture substrate or layer that has anon-culture surface in gaseous communication with an exterior of theapparatus can be adapted to have a structured surface defining gaspermeable wells as described herein. Examples of such cell cultureapparatuses include T-flasks, TRIPLE-FLASK cell culture vessels (Nunc.,Intl.), HYPERFLASK cell culture vessels (Corning, Inc.), CELLSTACKculture chambers (Corning, Inc.), CELLCUBE modules (Corning, Inc.), CELLFACTORY culture apparatuses (Nunc, Intl.), and cell culture articles asdescribed in WO 2007/015770, U.S. Patent Application Publication No.2014/0315296, U.S. Pat. No. 8,846,399, U.S. Pat. No. 8,178,345, and U.S.Pat. No. 7,745,209, which patents and published patent applications arehereby incorporated herein by reference in their respective entiretiesto the extent that they do not conflict with the disclosure presentedherein.

In some embodiments, gas-permeable/liquid impermeable materials are usedin construction of cell culture devices herein, or portions thereof(e.g., wells, microstructures, etc.) Any suitable gas-permeable/liquidimpermeable materials may find use, such as polystyrene, polycarbonate,ethylene vinyl acetate, polysulfone, polymethyl pentene (PMP),polytetrafluoroethylene (PTFE) or compatible fluoropolymer, a siliconerubber or copolymer, poly(styrene-butadiene-styrene), or polyolefin,such as polyethylene or polypropylene, or combinations of thesematerials. Substrate may be formed of any suitable material having asuitable gas permeability over at least a portion of the well. Examplesof suitable substrates include polydimethylsiloxane (PDMS),(poly)4-methylpentene (PMP), polyethylene (PE), and polystyrene (PS).PDMS can have a high degree of gas permeability and can be achievesufficient gas permeability at thicknesses up to 40 mm. PMP can achievesufficient gas permeability at thicknesses up to about 01 mm. In someembodiments, PMP having a thickness in a range of about 0.02 to 1 mm. PEor PS can achieve sufficient gas permeabilities at thicknesses up to 0.2mm, though thinner substrates may not have sufficient structuralintegrity. To compensate for poor structural integrity, an open frame,standoffs, or the like can be used to support the substrate from thebottom. In embodiments, a well thickness may be 0.02, 0.05, 0.1, 0.2,0.5, 1, 2, 5, 10, 20 or 40 mm, including ranges between any two of theforegoing. In embodiments, the wells have an oxygen transmission ratethrough the gas permeable polymeric material of 2000 cc/m²/day orgreater. In some embodiments, the wells have a gas permeability throughthe substrate of 3000 cc/m²/day or greater. In some embodiments, thewells have a gas permeability through the substrate of 5000 cc/m²/day orgreater.

Such materials allow effective gas exchange between outside and theinternal compartments, to allow the ingress of the oxygen and othergases, while preventing the passage of liquid or contaminants.

In some embodiments, the thickness of well substrate material isadjusted to allow for optimized gas exchange. In some embodiments,well-thickness is between 10 and 100 μm (e.g., 10 μm, 15 μm, 20 μm, 25μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100μm, and any ranges there between). Experiments conducted duringdevelopment of embodiments herein demonstrated higher numbers of viablecells produced with the thinner well-thickness (e.g., 17 μm>30 μm>57μm).

FIG. 8 is an illustration of an embodiment of the inclusion of asubstrate having an array of microwells, included as a surface of a cellculture device. In FIG. 8, the cell culture device is a flask 800.However, it should be understood that, in embodiments, the substrate mayform a part of any sort of cell culture device including, withoutlimitation, a multiwell plate, a flask, a dish, a tube, a multi-layercell culture flask, a bioreactor, or any other laboratory containerintended to grow cells or spheroids. The substrate having an array ofmicrowells may be gas permeable material. FIG. 9 shows a magnified viewof the substrate, shown at 9, micropatterned with an array ofmicrowells, forming the bottom surface of the flask shown in FIG. 8.

With reference to the embodiments illustrated in FIG. 8 and FIG. 9, Theillustrated apparatus is a flask or housing comprising a cell culturechamber 850. The housing includes a substrate having an array ofmicrowells (shown in FIG. 9). Housing also has a top surface 815, one ormore side walls 820 extending from the structured surface 110 to the topsurface 815. In some embodiments, the housing 850 includes a singleenclosing sidewall, such as a cylindrical wall or the like. Additionalexamples of such apparatuses are described in, for example,commonly-assigned U.S. provisional patent application Ser. No.62/072,015, which provisional patent application is incorporated hereinby reference in its entirety to the extent that it does not conflictwith the present disclosure.

The housing includes a port 860. Shown in FIG. 8 is an opening having ascrew top 861. However, in embodiments, the housing may have any type ofport to allow liquid and cells to enter and exit the housing. The port860 may be in a side wall, in the top surface 815 or in the cell culturesurface 110. Port 860 may connect to tubing or other connections tointroduce or remove cells and cell culture medium into the cell culturechamber 850.

While the housing depicted in FIG. 8 illustrates fixed sidewalls 820, inembodiments, the sidewalls may be flexible or expandable and collapsibleto allow a variable volume of cell culture medium into the cell culturechamber 850. As additional cell culture medium is introduced into thecell culture chamber 850, via port 860, flexible sidewalls 820 mayextend, and as cell culture medium is removed from the cell culturechamber 850, via port 860, flexible sidewall 820 may collapse. In someembodiments, sidewall 820 and top 815 are formed from a bag. Inaddition, cell culture chamber 850 can be filled with any volume of cellculture medium up to the fixed volume of the housing. In someembodiments (not shown) the entire or nearly the entire interior volumeis filled with cell culture medium.

In the embodiment depicted in FIG. 8, the volume of cell culture mediumin the cell culture chamber 850 is present at a height H. of mediumabove the cells. As described above, the height H. of medium in ahousing as described herein above cultured cells can be higher thanwould be possible if the wells were not gas permeable. In someembodiments, the cell culture chamber 850 is filled to its capacity forpurposes of culturing cells within the apparatus.

FIG. 10A shows HT29 cellular spheroids 500 inside the microwells 110 ofa micropatterned T25 spheroid forming flask, as shown in FIG. 9. FIG.10B shows harvested spheroids 500 from a micropatterned T25 spheroidforming flask, both according to Example 2 described below. FIG. 11A andFIG. 11B are micrographs illustrating the wide range size distributionof 3D aggregates (human ESC cells in FIG. 11A and mouse ESC in FIG. 11B)formed on a NUNCLON SPHERA™ low binding surface, available from Nunc.The NUNCLON SPHERA™ low binding surface has a low cell binding surfacetreatment, but lacks the geometry disclosed herein to allow for theformation of uniform spheroids

FIG. 12 is an illustration of a method of making a multiwell arraysubstrate, according to embodiments, and as described in Example 1below. While FIG. 12 shows a hot embossing/thermoforming process, othermethods of manufacturing microwell arrays according to embodiments arecontemplated, including coining, injection molding, embossing and othermethods known in the art.

FIG. 13, FIG. 14A and FIG. 14B, and FIG. 15 are graphs comparing viablecell count (FIG. 13 and FIG. 14A) and cell productivity (FIG. 14B andFIG. 15) for substrates having arrays of microwells versus flatsurfaces. The gas permeability of the wells may depend in part on thematerial of the substrate and the thickness of the substrate along thewell. In embodiments, the thickness of the sidewalls and bottom of wellsin a substrate having a microarray of wells may be constant and may berelatively thin. Or in embodiments, the walls of the wells in an arrayof microwells may be relatively thicker proximate to the opening intothe well, and relatively thinner at the bottom of the well. Or, inembodiments, walls of the wells in an array of microwells may berelatively thinner proximate to the opening into the well, andrelatively thicker at the bottom of the well. Depending on the materialused and the thickness employed, substrates having an array ofmicrowells may be gas permeable for purposes of the present disclosure.

FIG. 15 shows a graph depicting total protein titer excreted from MH677cells cultured on substrates having arrays of microwells versus flatsurface. These data are discussed below in Example 2.

While the apparatus depicted in FIG. 8 may be a hard-sided flask or asoft-sided cell culture flask, it will be understood that any other cellculture apparatus that includes a structured microwell array that has asurface that defines an exterior surface of the cell culture apparatusor that is in gaseous communication with an exterior of the apparatusmay have a substrate having an array of microwells formed from gaspermeable material as described herein.

A structured surface of a cell culture apparatus having an array ofmicrowells as described herein may define any suitable number of wellsthat may have any suitable size or shape. The wells define a volumebased on their size and shape. In many embodiments, one or more or allof the wells are symmetric and/or symmetrically rotatable around alongitudinal axis. In some embodiments, the longitudinal axes of one ormore or all of the wells are parallel with one another. The wells may beuniformly or non-uniformly spaced. In some embodiments, the wells areuniformly spaced. One or more or all the wells can have the same sizeand shape or can have different sizes and shapes.

In some embodiments, the thickness and shape of the substrate around thewell is configured to correct for refraction of light passing into theinterior surface and out of the exterior surface. In some embodiments,the correction is achieved by adjusting the thickness of the substratematerial forming the well. In some embodiments, the thickness of thesubstrate material proximate to the well-bottom (or nadir) is greaterthan the thickness of the substrate material in the sidewalls and/orproximate to the top aperture. In some embodiments, the thickness of thesubstrate material gradually decreases from a maximum at the nadir ofthe well-bottom to a minimum at the top aperture. For example, the shapeand thickness may be as described in commonly-assigned U.S. provisionalpatent application No. 62/072,019, which provisional patent applicationis hereby incorporated herein by reference in its entirety to the extentthat it does not conflict with the present disclosure.

The combination of, for example, non-adherent wells, spheroid inducingwell geometry, and gravity can define a confinement volume in whichgrowth of cells cultured in the wells is limited, which results in theformation of spheroids having dimensions defined by the confinementvolume.

In some embodiments, the inner surface of the wells 2115 is non-adherentto cells. The wells may be formed from non-adherent material or may becoated with non-adherent material to form a non-adherent well. Examplesof non-adherent material include perfluorinated polymers, olefins, orlike polymers or mixtures thereof. Other examples include agarose,non-ionic hydrogels such as polyacrylamides, polyethers such aspolyethylene oxide and polyols such as polyvinyl alcohol, or likematerials or mixtures thereof. The combination of, for example,non-adherent wells, well geometry, and gravity can induce cells culturedin the wells to self-assembly into spheroids. Some spheroids canmaintain differentiated cell function indicative of a more in vivo likeresponse relative to cells grown in a monolayer.

In some embodiments, one or more wells have a concave surface, such as ahemi-spherical surface, a conical surface having a rounded bottom, andthe like surface geometries or a combination thereof. The well and wellbottom can ultimately terminate, end, or bottom-out in a spheroidconducive rounded or curved surface, such as a dimple, a pit, and likeconcave frusto-conical relief surfaces, or combinations thereof. Othershapes and construction of gas-permeable spheroid-conducive wells aredescribed in commonly-assigned U.S. patent application Ser. No.14/087,906, which application is hereby incorporated herein by referencein its entirety to the extent that it does not conflict with the presentdisclosure.

In some embodiments, well bottoms are flat or come to a point. Wellbottoms may have any other suitable shape or dimension.

In some embodiments, the wells 115 described herein have a diametricdimension w in a range from about 200 micrometers to about 500micrometers, e.g., 200, 250, 300, 350, 400, 450 or 500 micrometers,including ranges between any two of the foregoing values. Such diametricdimensions can control the size of a spheroid grown therein such thatcells at the interior of the spheroid are maintained in a healthy state.In some embodiments, the wells 115 have a height H in a range from about100 micrometers to about 500 micrometers, e.g., 100, 150, 200, 250, 300,350, 400, 450 or 500 micrometers, including ranges between any two ofthe foregoing values. Of course, other suitable dimensions may also beemployed.

In some embodiments, the structured surface defining the wells includesan array of hexagonal close-packed well structures. An image of anembodiment of such a substrate having an array of hexagonal microwells100 is shown in FIG. 16, showing the substrate having an array ofhexagonal wells 1601. FIG. 17 is a schematic drawing showing cells(spheroids) 500 grown in wells 1601 of an embodiment of a substratehaving an array of microwells 100 having a hexagonal close-packed wellstructure. In some embodiments, the cells within each well 1601 form asingle spheroid 500, as depicted.

FIG. 18 is a side view illustrating an embodiment of a cell cultureapparatus including a porous membrane support. Various embodiments ofcell culture apparatuses 2100 incorporating a porous membrane support2500 are depicted in FIG. 18, FIG. 19 and FIG. 20. The porous membranesupport 2500 is disposed across the housing to the apparatus (e.g.,coupled to one or more sidewalls 2120) to compartmentalize the interiorof the housing into separate culture chambers 2152 and 2154. A firstculture chamber 2152 includes the substrate forming the structuredsurface defining gas permeable wells 2200 in gaseous communication withan exterior of the apparatus. The top of chamber 2152 is defined byporous membrane 2500. The bottom of the second culture chamber 2154 isdefined by porous membrane 2500. Accordingly, a first population ofcells 2200 can be cultured in the first chamber 2152 in wells 2115 ofthe structured surface formed by substrate 2110, and a second populationof cells 2202 can be cultured in second chamber 2154 on porous membrane2500.

The apparatuses depicted in FIG. 18, FIG. 19 and FIG. 20 include a firstport 2162 in communication with the first chamber 2152 and a second port2164 in communication with the second chamber 2154. In additionalembodiments, the first chamber 2152 or the second chamber 2154 mayoptionally have an additional port (an exit port, not shown) to allowfor flow of liquid through the chambers. The ports 2162, 2164 may beports similar to ports (e.g., ports 2160) depicted in, and discussedwith regard to, for example, FIG. 21, FIG. 22 and FIG. 23 below. Ports2162, 2164 can be on the same side of the apparatus 2100, as depicted,can be on opposing sides, or can be oriented in any other suitablemanner for providing separate access to the chambers or flow through thechambers 2152, 2154.

In FIG. 18, the apparatus is depicted as being operated with noheadspace (filled to capacity with cell culture medium). In FIG. 19 andFIG. 20, the apparatuses are depicted as being operated with headspace(not filled to capacity with culture medium). Due to the porous natureof support 2500, chamber 2152 remains filled with culture medium whilechamber 2154 can be operated with or without headspace. Due to the gaspermeable nature of the wells 2115, height of medium above the cells2200 may not be of a significant concern, e.g., as discussed above.However, if the housing is not otherwise gas permeable, it may bedesirable to limit the height of medium above cells cultured on porousmembrane support 2500. As depicted in FIG. 20, the porous membranesupport 2500 can form a substrate having an array of microwells, e.g.,as described above.

According to the embodiments shown in FIG. 18, FIG. 19 and FIG. 20,co-culture of more than one population of cells is contemplated. Forexample, a first population of cells may reside in the first chamber2152 while a second population of cells may reside in the second chamber2154. These populations of cells may be separated by a permeablemembrane 2500. This allows for chemical communication between a firstpopulation of cells and a second population of cells. In embodiments oneor both of these populations of cells may be spheroids. For example, asshown in FIG. 20, a first population of spheroid cells 2200 may grow inthe first chamber 2152, forming spheroids due to the spheroid inducinggeometry of the cell culture substrate, while a second population ofspheroid cells 2202, also forming spheroids due to the spheroid inducinggeometry of the cell culture substrate present in the second cellculture chamber, may grow in the second chamber 2154. Through the porousmembrane 2500, the second population of spheroid cells 2202 is inchemical communication with the first population of spheroid cells 2200.In this way, it is possible to co-culture two separate populations ofspheroid cells, while allowing the two separate populations of spheroidcells to be in chemical communication with each other. Or, inembodiments, as shown in FIG. 18, a first population of cells may bespheroid cells growing in a first cell culture chamber 2152 and a secondpopulation of cells that are not spheroid cells (because the second cellculture chamber does not have spheroid inducing geometry) may grow in asecond cell culture chamber 2154, while the presence of a porousmembrane 2500 allows the first and second populations of cells to be inchemical communication with each other. As shown in FIG. 19, the secondpopulation of cells may be grown in the presence of a head space, or asshown in FIG. 18, the second population of cells may be grown in theabsence of a head space. Similarly, a head space may be present orabsent from the first cell culture chamber. Or, spheroid inducinggeometry may be present or absent in the first cell culture chamber.Those of ordinary skill in the art will recognize that many combinationsof these features may be desirable, depending upon the cell culturerequirements of the user.

In some embodiments, the housing of an apparatus is gas-permeable. Byway of example a gas-permeable film or bag may form at least a portionof the housing. In some embodiments, one or more of the wells areconfigured, based at least in part upon their defined size and shape, togrow a single spheroid of a defined size. Spheroids may expand to sizelimits imposed by the geometry of the wells in which they are cultured.For example, each well may include a microwell or cell culture volumethat allows the spheroid to grow to a certain diameter. In other words,the geometrical dimensions of the microwell or cell culture volume mayconstrain the spheroid growth such that the spheroid diameter reaches amaximum value and stays at that maximum value. The production ofconsistently sized spheroids may lead to tissue-like, non-expandingspheroids that may be ideal for improving reproducibility of assayresults. The production of consistent spheroids may be the result of avariety of shaped and dimensioned volumes (e.g., a microwell or cellculture volume) defined by the interior of the one or more wells. Forexample, the microwell or cell culture volume may have a diametricdimension in a range from about 100 micrometers to about 700micrometers, such as from about 200 micrometers to 500 micrometers orany range within the aforementioned values (e.g., from 100 micrometersto 200 micrometers, from 100 micrometers to 500 micrometers, or from 200micrometers to 700 micrometers). The microwell or cell culture volumemay have a depth in a range from about 50 micrometers to about 700micrometers, such as about 100 micrometers to 500 micrometers, or anyrange within the aforementioned values.

FIG. 19 is a side view illustrating an additional embodiment of a cellculture apparatus including a porous membrane support.

FIG. 20 is a side view illustrating an additional embodiment of a cellculture apparatus including a porous membrane support illustratingco-culture of cells.

With reference to FIG. 21, an embodiment of cell culture apparatus 1400having a plurality of stacked cell culture compartments 1410A, 1410B,1410C is shown. Each cell culture compartment can include a substratehaving an array of microwells as described herein. Apparatus 1400includes a fill manifold 1430 having an opening 1435 through which cellculture media can be introduced or removed. Fill manifold 1430 includesa plurality of apertures (not shown). Each cell culture compartment1410A, 1410B, 1410C has at least one aperture (not shown) in fluidcommunication with an aperture of manifold 1430 such that cell culturemedia introduced through opening 1435 can flow into cell culturecompartments 1410A, 1410B, 1410C. Opening 1435 can be covered with a cap(not shown) or the like when apparatus 1400 is oriented in a cellculture position. Apparatus 1400 also optionally includes a ventmanifold 1420 defining an opening 1425 through which air, metabolicgases, and the like can flow. Vent manifold 1420 includes a plurality ofapertures (not shown). Each cell culture compartment 1410A, 1410B, 1410Chas at least one aperture (not shown) in gaseous communication with anaperture of manifold 1420 such that cell culture metabolic gases can beexchanged between an interior of a cell culture chamber and an exteriorof the apparatus 1400 via opening 1425. Opening 1425 can be covered witha vented cap (not shown), filter (not shown) or the like when apparatus1400 is oriented in a cell culture position.

Referring now to FIG. 22, a cross-sectional view of a cell cultureapparatus 1400, which can be a type of apparatus depicted in FIG. 21, isshown. Apparatus 1400 has a plurality of stacked cell culturecompartments 1410A, 1410B, 1410C, each having a substrate 1110 defininga structured surface having an array of gas permeable wells as describedabove. In the embodiment depicted in FIG. 22, each compartment (e.g.,1410B, 1410C), except for the top-most compartment 1410A in the stack,has a top surface 1450 defined by an second major surface of thestructured of substrate 1110 of an adjacent compartment. For example,second major surface of substrate 1110 of compartment 1410B serves asthe top interior surface of compartment 1410C. Accordingly, theinteriors of adjacent compartments are in gaseous communication witheach other through the common substrate 1110 that forms the bottomstructured surface/top surface. The top-most compartment 1410 has a topinner surface formed by top 1450, which can be a plate.

The interiors of the compartments (e.g., compartments 1410A, 1410B,1410C) are defined by a substrate having an array of microwells (e.g.,array 100 depicted in FIG. 9 a top surface defined by exterior secondmajor surface of the substrate of the compartment above, and one or moresidewalls 1440. The one or more side wall 1440 has an vent aperture 1442in communication with a vent column 1429 defined by manifold 1420, whichis in communication with an exterior of the apparatus via one or moreopenings 1425, 1426 defined by manifold 1420. Vent aperture 1442 definesa maximum volume of cell culture medium 1300 that can be present in theinterior of a cell culture chamber when the apparatus is a cell cultureorientation (e.g., as depicted in FIG. 22). The volume of cell culturemedium in the compartment can be less than the maximum. Vent aperture1442 also defines a minimum headspace volume in the interior of a cellculture compartment. The volume of headspace in the interior of thecompartment can be greater than the minimum headspace volume (if thecell culture medium volume is less than the maximum medium volume).

Accordingly, cells cultured in wells of a structured surface defined bysubstrate 1110 of a chamber (e.g., chamber 1410B) above an adjacentchamber (e.g., chamber 1410C) are in gaseous communication with theheadspace 1441 of the adjacent chamber (e.g., chamber 1410C) via the gaspermeable wells. Headspace 1441 is in communication column 1429 definedby manifold, which is in communication with an exterior of the apparatusvia one or more openings 1425, 1426.

An optional filter 1427 can be incorporated into the opening 1425,opening 1425, or in cap 1422 to vent metabolic gases. Having a vent onthe bottom of the apparatus can be advantageous in some embodiments. Forexample, the metabolic waste gas carbon dioxide is more dense thanatmospheric air and tends to form a gradient with highest concentrationson the bottom in culture apparatuses that do not have a bottom vent.Accordingly, the presence of a vent on the bottom of the apparatus(e.g., formed by vent column 1429 and opening 1426) can facilitatetransfer of waste carbon dioxide out of the apparatus.

Referring now to FIG. 23, a cross-sectional view of a cell cultureapparatus 1400, which can be a type of apparatus depicted in FIG. 21 andmay be a portion of the apparatus depicted in FIG. 22, is shown. To theextent that each reference numeral in FIG. 23 is not explicitlydiscussed, reference is made to the discussion of like numberedcomponents described above with regard to FIG. 22. In the illustratedembodiment, interiors of the compartments (e.g., compartments 1410A,1410B, 1410C) are defined by an interior structured surface of substrate1110, a top surface defined by an exterior surface of the substrate ofthe compartment above, and one or more sidewalls 1440. The one or moreside walls 1440 have an aperture 1443 in communication with column 1439defined by fill manifold 1430, which is in communication with anexterior of the apparatus via opening 1435 defined by manifold 1430,which may be covered by a cap 1432 when the apparatus is in a cellculture orientation. Cell culture medium 1300 can be introduced intocell culture compartments (e.g., compartments 1410A, 1410B, 1410C) orremoved from cell culture compartments via column 1439 and opening 1435via manipulation of the apparatus.

Referring now to FIGS. 24A and 24B, a schematic bottom view (FIG. 24A)and a schematic perspective view (FIG. 24B) are shown of a tray 1415that can serve to form cell culture compartments (e.g., compartments1410A, 1410B, 1410C as depicted in FIG. 21, FIG. 22 and FIG. 23) when aplurality of such trays are stacked on top of one another. The tray 1415includes sidewalls 1440A, 1440B, 1440C, 1440D that extend from asubstrate 1110 defining a substrate having an array of microwells. Insome embodiments, tray includes a single enclosing sidewall (not shown).A partial height wall 1472 is coupled to sidewall 1440A and sidewall1440D. Partial height wall 1472 and sidewalls 1440A, 1440D surround anddefine vent aperture 1442. When a cell culture apparatus is assembledfrom stacked trays, metabolic gases within an interior of a chamberformed by tray 1415 can flow over partial wall 1472 and through aperture1442. Vent apertures 1442, partial walls 1472 and associated sidewallsof stacked trays 1415 can form at least a portion of a vent column(e.g., vent column 1429 depicted in FIG. 22).

Tray 1415 also includes a partial height wall 1473 coupled to sidewall1440A and to sidewall 1440B. Partial height wall 1473 and sidewalls1440A, 1440B surround and define fill aperture 1443. When a cell cultureapparatus is assembled from stacked trays, culture media can beintroduced to or removed from an interior of a chamber formed by tray1415 over partial wall 1473 and through fill aperture 1443 viamanipulation of the assembled apparatus. Fill apertures 1443, partialwalls 1473 and associated sidewalls of stacked trays 1415 can form atleast a portion of a fill column (e.g., fill column 1439 depicted inFIG. 23).

The height of partial wall 1473 defines a maximum height and volume ofcell culture medium that can be contained within a cell culturecompartment formed by tray 1415. The top of the partial wall 1473 can beany suitable distance from the substrate 1110 forming the structuredsurface. In some embodiments, the distance is 5 mm or greater, such as6, 7, 8, 9, 10, 12, 15, or 20 mm, including ranges between any two ofthe foregoing. Because of the gas permeable wells of the structuredsurfaces of cell culture apparatuses assembled from such trays are incommunication with an exterior of the apparatus, the height of the cellculture medium above the cells can be greater than available withconventional cell culture apparatuses in which gas exchange occursprimarily through the cell culture medium.

The distance from the top of partial wall 1472 to the substrate 1110 canbe the same or greater than the distance from the top of partial wall1473 to the substrate 1110. Accordingly, if the compartment isoverfilled with culture medium, excess medium will drain through fillaperture 1443 rather than vent aperture 1442. In embodiments employing abottom filter (such as filter 1427 in FIG. 22) in a vent column, thebottom filter will not be contaminated with medium. Of course, propermanipulation of an assembled apparatus should also prevent the bottomfilter from being contaminated with medium.

Referring now to FIG. 25, a schematic side view of an embodiment of acell culture apparatus 1400 having a plurality of stacked cell culturecompartments 1410A, 1410B, 1410C is shown. Each compartment can includea substrate 1110 defining a structured surface as described above.Apparatus 1400 includes spacers 1500 positioned adjacent the substrate1110 forming the structured surface and, exterior to the chamber,providing a passageway for air flow, which passage way is referred toherein as a “tracheal space” (e.g., tracheal spaces 1460A, 1460B,1460C). Because the structured surface defines gas permeable wells inwhich cells can be cultured, metabolic gases may be exchanged throughthe wells to a tracheal space 1460A, 1460B, 1460C defined by spacers1500 to an exterior of the apparatus 1400. Apparatus 1400 also includesa manifold 1430 defining an opening through which cell culture media canbe introduced or removed. Manifold 1430 includes a plurality ofapertures (not shown). Each cell culture compartment 1410A, 1410B, 1410Chas at least one aperture (not shown) in fluid communication with anaperture of manifold 1430 such that cell culture media introducedthrough manifold 1430 can flow into cell culture compartments 1410A,1410B, 1410C.

The bottom of the cell culture apparatus 1400 in FIG. 25 includes aplate 1510 on which spacers 1500 are disposed. In embodiments, the plate1510 and spacers 1500 are a singled part, such as a molded part. Such aplate may form the top surface of the other cell culture compartments(e.g., compartments 1410A, 1410B, 1410C). For each compartment, one ormore sidewall 1440 extends from the substrate 1100 defining thestructured surface to the top surface, which may be formed of a platewith spacers. Aperture (not shown) in communication with an aperture(not shown) of port 1430 can be defined by a sidewall.

Stacked cell culture trays or chamber can be assembled in any suitablemanner. For example such components can be joined using weldingtechniques (e.g., thermal, laser, long IR or ultrasonic welding, or thelike), adhering, solvent-bonding or the like

In some embodiments, the structured surface is coupled to a bag. Bagssuitable for cell culture can be formed from films by heat sealing,laser welding, application of adhesive, or any other method known in theart of inflatable bag making. Walls or portions thereof of a bag mayhave a thickness that allows for efficient transfer of gas across thewall. It will be understood that desired thickness may vary depending onthe material from which the wall is formed. By way of example, the wallor film forming the wall may be between about 0.02 millimeters and 0.8millimeters thick. A bag may be made of any material suitable forculturing cells. In various embodiments, the bag is formed of opticallytransparent material to allow visual inspection of cells cultured in thebag. Examples of optically transparent, gas permeable materials that maybe used to form the bag include polystyrene, polycarbonate,poly(ethylene vinyl acetate), polysulfone, polymethylpentene,polytetrafluoroethylene (PTFE) or compatible fluoropolymer, a siliconerubber or copolymer, poly(styrene-butadiene-styrene), or polyolefin,such as polyethylene or polypropylene, or combinations of thesematerials.

The cell culture apparatuses described herein can be used to culturecells within wells of a structured surface. As described above, the cellculture medium in an apparatus can be any suitable height above thecells. In some embodiments, the height of the cell culture medium in theapparatus above the cells (e.g., above the top or the nadir of the wellsis about 5 mm or greater. In some embodiments, the height of the cellculture medium in the apparatus above the cells (e.g., above the top orthe nadir of the wells is about 6 mm or greater, about 7 mm or greater,about 8 mm or greater, about 9 mm or greater or about 10 mm or greater.

Because of the gas permeability of the wells, such heights of cellculture medium can be used to maintain the cells in a healthy state.Accordingly, cells cultured in the apparatuses described herein can becultured for extended periods of time with such heights of cell culturemedium. For example, the cells can be continuously cultured with suchmedia heights for 24 hour or longer, for 48 hours or longer, for 72hours or longer, for 96 hours or longer, or until the medium isexchanged.

In embodiments where the wells are non-adherent to cells, the cells maybe harvested by inverting the apparatus to allow gravity to displace thecells from the wells.

In some embodiments, a porous membrane is disposed in a cell cultureapparatus as described herein to support growth of a second cell-typewithin the same apparatus, but separated from a first cell type culturedon the structured surface in gaseous communication with the exterior ofthe apparatus (or to support growth of additional cells of the sametype). In some embodiments, stem cells are cultured on the structuresurface having the gas permeable wells in communication with an exteriorof the apparatus and feeder cells are cultured on the porous membrane.Of course, any other desired combination of cells andcompartmentalization can be employed using such an apparatus.

The porous membrane can be disposed within a housing of a cell cultureapparatus to compartmentalize the housing into two growth chambers.Preferably the permeable membrane limits cell movement through themembrane but permits passage of biomolecules. Examples of materials thatcan be used to form a porous membrane include track-etched membranes orwoven or non-woven porous materials. The material of the porous membranemay be treated or coated to make it more adherent or more non-adherentto cells. Treatment may be accomplished by any number of methods knownin the art which include plasma discharge, corona discharge, gas plasmadischarge, ion bombardment, ionizing radiation, and high intensity UVlight. Coatings can be introduced by any suitable method known in theart including printing, spraying, condensation, radiant energy,ionization techniques or dipping. The coatings may then provide eithercovalent or non-covalent attachment sites. Such sites can be used toattach moieties, such as cell culture components (e.g., proteins thatfacilitate growth or adhesion). Further, the coatings may also be usedto enhance the attachment of cells (e.g., polylysine). Alternatively,cell non-adherent coatings as described above can be used to prevent orinhibit cell binding. In some embodiments, the porous membrane can befabricated to have a structured surface having a plurality of wells,such as described above with regard to the substrate forming thestructured surface defining the plurality of gas permeable wells.However, in this case, the porous membrane material is formed to havethe structured surface.

The gas permeable wells of the structured surface (e.g., as describedabove) permits control of oxygen tension by regulating gas concentrationin an incubator where the apparatus is placed to permit cell growth. Thepermeable membrane support provides a method to physically separatedifferent cell populations while permitting transfer of biologicallyactive components.

The porous membrane may be attached to housing (e.g., sidewalls, etc.)of the apparatus in any suitable manner. For example, the porousmembrane can be incorporated into the device in a similar manner toincorporation of the substrate forming the substrate defining thestructured surface having the plurality of gas permeable wells, asdescribed above.

In the embodiment depicted in FIG. 26, the cell culture apparatus 3100is a 96-well multiwell plate having wells 3115 in a plate 3111surrounded by a frame 3113. However, as discussed above, a cell cultureapparatus can have any suitable number of wells (for example, 3, 6, 12,96, or any other number of wells may be provided). In the depictedembodiment, at least a portion of each of the plurality of wells 3115contains a substrate having an array of microwells and provides aposition to form spheroids, as described above. Nearly any type of cellculture apparatus having a well that is used to culture cells may bedesigned by employing a substrate having an array of microwells whichcan, in some embodiments, form the entire volume of the well or can, insome embodiments, form a cell culturing sub-volume of the well. In someembodiments, the cell culture apparatuses with which the microwelldesign may be implemented may be a multi-well plate, for example, a96-well multiwell plate, a 384-well multiwell plate, a 1536-wellmultiwell plate, or the like. In some embodiments, at least a surface ofthe multiwell plate is gas-permeable.

The ability for the plurality of wells to allow cells to aggregate insuch a way that spheroids form as well as the ability for the spheroidsformed within each of the plurality of wells to maintain a consistentsize across the plurality of wells can be accomplished in any suitablemanner. For example, the plurality of wells 3115 may comprise an arrayof microwells structured and arranged like the microwells shown in FIG.1A, FIG. 1B, FIG. 2A, FIG. 2B, FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D, FIG.3E, FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, FIG. 5A, FIG. 5B, FIG. 5C, FIG.5D, FIG. 5E, or FIG. 16, in which cells grow as spheroids 500.

In some embodiments, one or more of the wells are configured, based atleast in part upon their defined size and shape, to grow a singlespheroid of a defined size. Spheroids may expand to size limits imposedby the geometry of the wells in which they are cultured. For example,each well may include a microwell or cell culture volume that allows thespheroid to grow to a certain diameter. In other words, the geometricaldimensions of the microwell or cell culture volume may constrain thespheroid growth such that the spheroid diameter reaches a maximum valueand stays at that maximum value. The production of consistently sizedspheroids may lead to tissue-like, non-expanding spheroids that may beideal for improving reproducibility of assay results. The production ofconsistent spheroids may be the result of a variety of shaped anddimensioned volumes (e.g., a microwell or cell culture volume) definedby the interior of the one or more wells. For example, the microwell orcell culture volume may have a diametric dimension in a range from about100 micrometers to about 700 micrometers, such as from about 200micrometers to 500 micrometers or any range within the aforementionedvalues (e.g., from 100 micrometers to 200 micrometers, from 100micrometers to 500 micrometers, or from 200 micrometers to 700micrometers). The microwell or cell culture volume may have a depth in arange from about 50 micrometers to about 700 micrometers, such as about100 micrometers to 500 micrometers, or any range within theaforementioned values.

Each of the plurality of wells described herein may assist cellsdeposited therein to form spheroids. Each of the plurality of wells mayalso limit or constrain a diameter of each of the spheroids to a valueof about, e.g., less than or equal to 500 micrometers, less than orequal to 400 micrometers, less than or equal to 300 micrometers, lessthan or equal to 250 micrometers, less than or equal to 150 micrometers,etc. or any range within the aforementioned values (e.g., 150 to 250,150 to 300, 150 to 400, 150 to 500, 250 to 300, 250 to 400, etc.). Insome embodiments, each of the plurality of wells forms a spheroiddefined by a diameter that differs from an average diameter of all thespheroids grown in the plurality of wells by about, e.g., less than orequal to 20%, less than or equal to 15%, less than or equal to 10%, lessthan or equal to 5%, less than or equal to 2%, etc. or any range withinthe aforementioned values.

In embodiments shown in FIG. 27, FIG. 28 and FIG. 29, the array ofmicrowells 3115 are formed in a substrate 3110. Each of the plurality ofwells 3115 may have a top aperture 3118, a bottom surface 3112, and asidewall surface 3120 extending from the top aperture 3118 to the bottomsurface 3112. Additionally, the sidewall surface 3120 may define a pentip area 3116 (see FIG. 29) between the top aperture 3118 and the bottomsurface 3112, so named because the constricted area at the bottom of thewell looks like the tip of a pen. The pen tip area is aspheroid-inducing geometry.

The top aperture 3118 of each of the plurality of wells 3115 may be usedas an opening through which to seed cells into each of the plurality ofwells 3115. The top aperture 3118 may have a variety of different shapesand sizes. For example, the top aperture 3118 may be defined by a shapethat is circular, oval, square, rectangular, hexagonal, quadrilateral,etc. The top aperture 3118 may also be defined by a diametric dimension(e.g., diameter, width, etc., depending on shape). The diametricdimension of the top aperture 3118 may be defined as a distance acrossthe top aperture at a widest point. The diametric dimension of the topaperture 3118 may be about, e.g., greater than or equal to 300micrometers, greater than or equal to 500 micrometers, greater than orequal to 800 micrometers, greater than or equal to 1000 micrometers,greater than or equal to 1500 micrometers, greater than or equal to 2000micrometers, etc. or, less than or equal to 7000 micrometers, less thanor equal to 6000 micrometers, less than or equal to 4000 micrometers,less than or equal to 2500 micrometers, less than or equal to 1700micrometers, less than or equal to 1200 micrometers, etc. or any rangewithin the aforementioned values.

The bottom surface 3112 of each of the plurality of wells 3115 may beconducive to allowing cells to be cultured thereon or there-above. Thebottom surface 3112 may have a variety of different shapes and sizes.For example, the bottom surface 3112 may be rounded, hemispherical,flat, conical, etc. By way of further example, the bottom surface 3112may also be defined by a shape that is circular, oval, square,rectangular, hexagonal, quadrilateral, etc. As shown in FIGS. 27-29 thebottom surface 3112 is flat and is defined by a diametric dimension. Thediametric dimension of the bottom surface may be about, e.g., greaterthan or equal to 0 micrometers, greater than or equal to 50 micrometers,greater than or equal to 75 micrometers, greater than or equal to 100micrometers, greater than or equal to 200 micrometers, greater than orequal to 275 micrometers, etc. or, less than or equal to 700micrometers, less than or equal to 500 micrometers, less than or equalto 400 micrometers, less than or equal to 300 micrometers, less than orequal to 250 micrometers, less than or equal to 150 micrometers, etc. orany range within the aforementioned values. For bottom surfaces 3112that have a rounded bottom or similar surface (e.g., hemispherical,conical, etc.), with a nadir located at the lowest point, the diametricdimension of the bottom surface 112 is considered to be zero. In someembodiments, the diametric dimension of the top aperture 3118 is greaterthan the diametric dimension of the bottom surface 3112. In otherembodiments, the diametric dimension of the top aperture 3118 is equalto the diametric dimension of the bottom surface 3112.

In some embodiments, the bottom surface 3112 of each of the plurality ofwells 3115 may be uniformly constructed with the substrate having amicroarray of wells (see also FIG. 29). In other embodiments, the bottomsurface 3112 may be made from a material that is different from thematerial used to form substrate 3110. Various methods of manufacturingthe plurality of wells will be described further below. The bottomsurface 3112 or the sidewall surface 3120 may be gas permeable to helpprovide oxygen to the cells or spheroids 3130 cultured within the wells3115. In some embodiments, the substrate 3150 that defines the bottomsurface 3112 may be a gas permeable substrate. In some embodiments, thesubstrate 3150 may comprise a gas permeable film. The gas permeabilityof the bottom surface 3112 to an exterior will depend in part on thematerial of the bottom surface 3112 and the thickness of the bottomsurface 3112. For example, the gas permeability of the wells may be asdescribed in U.S. provisional patent application No. 62/072,088, filedon 29 Oct. 2014, and entitled “GAS PERMEABLE CULTURE FLASK,” whichprovisional patent application is hereby incorporated herein byreference in its entirety to the extent that it does not conflict withthe present disclosure.

The pen tip area 3116 (see FIG. 29) may be defined by the sidewallsurface 3120 between the top aperture 3118 and the bottom surface 3112.A location of the pen tip area 3116 may be defined by other componentsof the well. For example, the pen tip area 3116 may be defined by adiametric dimension 3144 across the sidewall surface 3120. The diametricdimension 3144 of the pen tip area 3116 may be defined as a distanceacross the sidewall surface 3120 at the pen tip area 3116. The pen tiparea 3116 may be defined by a diametric dimension 3144 of about, e.g.,greater than or equal to 50 micrometers, greater than or equal to 100micrometers, greater than or equal to 200 micrometers, greater than orequal to 300 micrometers, greater than or equal to 400 micrometers,greater than or equal to 550 micrometers, etc. or, less than or equal to800 micrometers, less than or equal to 700 micrometers, less than orequal to 600 micrometers, less than or equal to 500 micrometers, lessthan or equal to 450 micrometers, less than or equal to 350 micrometers,etc. or any range within the aforementioned values. The pen tip area3116 may also be defined by a height 3142 from the bottom surface 3112of about, e.g., greater than or equal to 50 micrometers, greater than orequal to 100 micrometers, greater than or equal to 150 micrometers,greater than or equal to 250 micrometers, greater than or equal to 350micrometers, greater than or equal to 450 micrometers, etc. or, lessthan or equal to 800 micrometers, less than or equal to 700 micrometers,less than or equal to 600 micrometers, less than or equal to 500micrometers, less than or equal to 400 micrometers, less than or equalto 300 micrometers, etc. or any range within the aforementioned values.The height 3142 may be measured from a lowest point of the bottomsurface 3112. In such embodiments, the entire volume of the well 3115 isthe cell culturing volume 3140.

The diametric dimension of the top aperture 3118 may be greater or equalto the diametric dimension 3144 of the pen tip area 3116. The diametricdimension 3144 of the pen tip area 3116 may be greater than or equal tothe diametric dimension of the bottom surface 3112. It may also bedescribed that the diametric dimension of the bottom surface 3112 isless than or equal to the diametric dimension 3144 of the pen tip area3116 or the diametric dimension 3144 of the pen tip area 3116 may beless or equal to the diametric dimension of the top aperture 3118. Insome embodiments, the top aperture 3118 may be the pen tip area 3116.

The sidewall surface 3120 of each of the plurality of wells 3120 extendsfrom the top aperture 3118 to the bottom surface 3112. The sidewallsurface 3120 may include an upper sidewall surface 3124 and a lowersidewall surface 3122. The upper sidewall surface 3124 may be definedbetween the top aperture 3118 and the pen tip area 3116. The lowersidewall surface 3122 may be defined between the pen tip area 3116 andthe bottom surface 3112. In some embodiments, the sidewall surface 3120of each of the plurality of wells 3115 may define a cell non-adherentsurface. The cell non-adherent surface facilitates growing the cellsinto spheroids 3130 in the cell culturing volume 3140 as describedpreviously. Cell non-adherent upper sidewall surfaces 3124 canfacilitate settling of seeded cells into the cell culture volume 3140.Regardless of whether the upper sidewall surfaces 3124 are cellnon-adherent, the upper sidewall surfaces 3124, in some embodiments, areconfigured to cause cells seeded in the wells to settle into the pen tiparea 3116 to form the cell culturing volume 3140 as a result of gravity.

In some embodiments, the upper sidewall surface 3124 and the lowersidewall surface 3122 may be defined by a shape that is, e.g.,parabolic, conical, stepped, various angles, curved, etc. The upper andlower sidewall surfaces 3124, 3122 may have the same or differentshapes. In some embodiments, the sidewall surface 3120 may have aninflection point 121 at a location where the upper and lower sidewallsurfaces 3124, 3122 meet (e.g., as shown in FIG. 29). In otherembodiments, the sidewall surface 3120 may have a continuous slope atthe inflection point 3121 where the upper and lower sidewall 3124, 3122surfaces meet.

In some embodiments, a portion of sidewall 3120 adjoining the bottomsurface 112 may be normal to the bottom surface 3112 or at an angle tothe bottom surface 3112. The portion of the sidewall 3120 adjoining thebottom surface 3112 may be described as the lower sidewall surface 3122.The angle at which the portion of the sidewall 3120 intersects thebottom surface 3112 may be defined relative to the bottom surface 3112as, e.g., greater than or equal to 90 degrees, greater than or equal to92 degrees, greater than or equal to 95 degrees, greater than or equalto 100 degrees, etc. or, less than or equal to 110 degrees, less than orequal to 105 degrees, less than or equal to 102 degrees, less than orequal to 97 degrees, etc. or any range within the aforementioned values.In some embodiments, the diametric dimension across the sidewall surface3120 may be described as increasing from the bottom surface 3112 towardsthe top aperture 3118. The sidewall geometry may be any geometry thatsufficiently allows the cells to settle into each of the plurality ofwells 3115.

With regards to a bottom surface 3112 that does not have a flat surface,the angle of the sidewall 3120 is considered to be relative to animaginary plane that is tangential to the nadir of the bottom surface3112. In other embodiments, the imaginary plane may also be defined asbeing coplanar with the top aperture 3118 regardless of whether theimaginary plane is tangential to the nadir.

A combination of the bottom surface 3112, the pen tip area 3116 and aportion of the sidewall surface 3120 may define a cell culture volume3140. The portion of the sidewall surface 3120 that defines the cellculture volume 3140 may also be described as the lower sidewall surface3122. The cells are not restricted to being cultured only in the cellculture volume 3140. However, cells deposited within each of theplurality of wells 3115 may aggregate in the cell culture volume 3140 toform and grow a spheroid 3130. Also, the dimensions of the spheroid 3130may be a result of the shape and size of the cell culture volume 3140.For example, the cell culture volume 3140 of each of the plurality ofwells 3115 is configured to cause spheroids 3130 to grow to a diameterof about, e.g., less than or equal to 500 micrometers, less than orequal to 400 micrometers, less than or equal to 300 micrometers, lessthan or equal to 250 micrometers, less than or equal to 150 micrometers,etc. or any range within the aforementioned values. In some embodiments,the cell culture volume 3140 of each of the plurality of wells 3115forms a spheroid 3130 defined by a diameter that differs from an averagediameter of all the spheroids 3130 grown in the plurality of wells 3115by about, e.g., less than or equal to 20%, less than or equal to 15%,less than or equal to 10%, less than or equal to 5%, less than or equalto 2%, etc. or any range within the aforementioned values.

A combination of the top aperture 3118, the pen tip area 3116 and aportion of the sidewall surface 3120 may define a second volume 3145.The portion of the sidewall surface 3120 that defines the second volume3145 may also be described as the upper sidewall surface 3124. Thesecond volume 3145 may be greater than the cell culture volume 3140. Forexample, the second volume 3145 may be about, greater than or equal to100%, greater than or equal to 200%, greater than or equal to 500%,greater than or equal to 1,000%, greater than or equal to 10,000%,greater than or equal to 100,000%, greater than or equal to 200,000%,etc. of the cell culture volume 3140 or any range within theaforementioned values. By way of example, a 96-well plate may have acell culture volume defined by a volume of 0.1 microliters and a secondvolume defined by a volume of 200 microliters, resulting in a secondvolume that is 2,000 times greater in volume than the cell culturevolume.

One embodiment of the cell culture apparatus 3100 is illustrated in FIG.27. As shown in FIG. 27, the sidewall surface 3120 of each of theplurality of wells 3115 tapers from the top aperture 3118 to the bottomsurface 3112. Specifically, the sidewall surface 3120 is extending fromthe top aperture 3118 in a direction that is almost normal to the topaperture 3118 but at a slight angle that decreases a diametric dimensionacross the sidewall surface 3120 as the sidewall surface 3120 extendstowards the bottom surface 3112. At a point 3123 along the sidewallsurface 3120 that is between the top aperture 3118 and the pen tip area3116, the angle of the sidewall surface 3120 changes to further decreasethe diametric dimension across the sidewall surface 3120 as the sidewallsurface 3120 extends towards the bottom surface 3112. At the pen tiparea 3116, the angle of the sidewall surface 3120 changes yet again andextends towards the bottom surface 3112. This last portion of thesidewall surface 3120 is sometimes described as the lower sidewallsurface 3122. As shown in FIG. 27, in embodiments, the lower sidewallsurface 3122 may be slightly angled from being perpendicular to thebottom surface 3112 and the diametric dimension across the sidewallsurface 3120 decreases as the sidewall surface 3120 extends towards thebottom surface 3112. A spheroid 3130 is depicted as positioned withinthe lower sidewall surface 3122 and against the bottom surface 3112(i.e. in the pen tip area 3116). The lower sidewall surface 3122 mayrestrict or limit the size to which the spheroid 3130 can grow.

One embodiment of the cell culture apparatus 3100 is illustrated in FIG.28. As shown in FIG. 28, the sidewall surface 3120 of each of theplurality of wells 3115 tapers from the top aperture 3118 to the bottomsurface 3112. Specifically, the sidewall surface 3120 initially extendsfrom the top aperture 3118 at an angle normal to the top aperture 3118and then extends along a parabolic path towards the pen tip area 3116.At the pen tip area 3116, the angle of the sidewall surface 3120 changesand extends towards the bottom surface 3112. This last portion of thesidewall surface 3120 is sometimes described as the lower sidewallsurface 3122. As shown in FIG. 29, the lower sidewall surface 3122 isslightly angled from being perpendicular to the bottom surface 3112 andthe diametric dimension across the sidewall surface 3120 decreases asthe sidewall surface 3120 extends towards the bottom surface 3112. Aspheroid 3130 is depicted as positioned within the lower sidewallsurface 3122 and against the bottom surface 3112, in the pen tip area3116 The lower sidewall surface 3122 may restrict or limit the size towhich the spheroid 3130 can grow. The pen tip area is a spheroidinducing geometry.

Turning now to FIG. 30, in some embodiments, the cell culture apparatus3650 may include a bottom plate 3610 and one or more sidewalls 3620, asshown in FIG. 30. The bottom plate 3610 may define a major surface 3611and the one or more sidewalls 3620 may extend from the bottom plate3610. The bottom plate 3610 may be formed, in whole or in part, from asubstrate having an array of microwells 3615. FIG. 30 illustrates thatthe bottom plate may have an array of arrays of microwells 3615. Thatis, each of the areas identified as an array of microwells 3615 shown inFIG. 30 may contain an array of much smaller microwells. In embodiments,cell culture apparatus 3650 may also include a plurality of wells 3615formed in the major surface 3611 of the bottom plate 3610. Each well ofthe plurality of arrays of microwells 3615 may define microwells or cellculture volumes, as described previously, that promote or induce thegrowth of spheroids. The major surface 3611 of the bottom plate 3610 andthe one or more sidewalls 3620 define a reservoir volume. Reservoirplates described herein permit the addition of culture medium in excessof what would be typically used to fill individual shallow wells of amicrowell plate and allows cells cultured in different wells to be influid communication.

In some embodiments, the one or more sidewalls 3620 may extend fartheraway (e.g., a sidewall height) from the bottom plate 3610 than somecurrently available cell culture apparatuses, allowing the reservoir tohold a larger than normal volume of medium. The larger capacityopportunity for the reservoir may allow an excess of culture medium tobe added to the reservoir so that the spheroids may not need to relyonly on the amount of medium in each individual well. In other words,the spheroids may not need to be fed with cell culture medium asfrequently as spheroids growing in standard microplate wells. As shownin FIG. 30, nutrients and metabolites may be exchanged throughout thecell culture medium because the cell culture medium in the reservoir isin communication with all of the wells in the reservoir.

In some embodiments, a cell culture assembly 3600 may include a cellculture apparatus 3650 and a fluid permeable mesh 3670. The fluidpermeable mesh 3670 can be placed on top of the wells 3615 after cellshave been seeded into the wells. The cell culture medium that is incommon communication among the plurality of wells 3615 can be isolatedand replaced during a manual batch feeding process without disturbingthe cells in the wells. Because the cells, in some embodiments, can benon-adherent to the surface of the wells, exchange of cell culture mediawithout disturbing or losing the spheroid can be difficult. However, useof mesh 3670 as discussed above can mitigate such difficulties. Forexample, the combination of a cell culture apparatus and fluid permeablemesh may be as described in U.S. provisional patent application No.62/072,103, filed on 29 Oct. 2014, and entitled “RESERVOIR SPHEROIDPLATE,” which provisional patent application is hereby incorporatedherein by reference in its entirety to the extent that it does notconflict with the present disclosure.

It will be understood that the wells 3615 of a cell culture apparatus3650 described herein can be of any size, shape or configuration. Insome embodiments, the wells are formed from hexagonal close-packed wellstructures as depicted in FIG. 17. A reservoir plate apparatus having astructured surface with closely packed small volume wells may beparticularly advantageous because the small volume of the wells wouldrequire frequent exchange of cell culture medium without the addedreservoir volume.

Reservoir plates described herein permit, for example, the addition ofculture medium in excess of what would be typically used to fillindividual shallow wells of a microwell plate and allows cells culturedin different wells to be in fluid communication.

As shown in FIG. 31, the cell culture apparatus 4100 may include abottom plate 4110 and one or more sidewalls 4120. The bottom plate maydefine a major surface and the one or more sidewalls 4120 may extendfrom the bottom plate. The combination of the bottom plate and the oneor more sidewalls may define a reservoir. The cell culture apparatus mayalso include, in whole or in part, substrates having an array ofmicrowells 4115 formed in the major surface of the bottom plate. Eachwell of the plurality of wells in the microwell array may define anupper aperture and a nadir. The upper aperture may be co-planar with themajor surface and the nadir may be positioned below the major surface,i.e., the nadir may be positioned a direction opposite that which theone or more sidewalls extend from the bottom plate. A top plate (notshown) may be disposed over the reservoir as desired while incubatingcells.

In some embodiments, the one or more sidewalls may extend farther awayfrom the bottom plate than typical, and therefore, allowing thereservoir to hold a larger than normal volume of medium. The largercapacity opportunity for the reservoir may allow an excess of culturemedium to be added to the reservoir so that the spheroids may not needto rely only on the amount of medium in each individual well. In otherwords, the spheroids may not need to be fed with cell culture medium asfrequently as spheroids growing in standard microplate wells. As shownin FIG. 31, nutrients and metabolites may be exchanged throughout thecell culture medium because the cell culture medium in the reservoir isin communication with all of the wells in the reservoir.

In some embodiments, a cell culture assembly 4200 is described herein.The assembly can include an apparatus 4100 (e.g., as depicted anddiscussed with regard to FIG. 31) and a fluid permeable mesh 4570. Thefluid permeable mesh 4570 can be placed on top of the wells 4115 aftercells have been seeded into the wells. The cell culture medium in commoncommunication can be isolated and replaced during a manual batch feedingprocess without disturbing the cells in the wells.

In some embodiments (e.g., as depicted in FIG. 32), a frame 4560 can becoupled to the mesh 4570, as shown. The frame 4560 may be configured tomaintain the mesh 4570 in place over the wells first well 4515. In someembodiments, the mesh 4570 is configured to be disposed over an upperedge of sidewall 4120 of apparatus 4100. The frame 4560 can engage oneor more sidewall 4120 via interference fit, snap fit, or any othersuitable mechanism to retain the mesh on the major surface of the plate4110. In some embodiments, a user can manually retain the frame 4560 inplace such that the mesh is retain on the major surface of the plate4110 over the wells 4115.

Fluid permeable mesh 4570 may be formed of any suitable material. Insome embodiments, fluid permeable mesh defines pores. The pores can beof any suitable size. In some embodiments the pores define an averagepore size in a range from 10 micrometers to 100 micrometers. In someembodiments, the pores define an average pore size of less than or equalto 40 micrometers. Preferably, the pores of the mesh are of asufficiently small size to prevent passage of a spheroid through themesh.

In some embodiments, mesh can be as described in, for example,commonly-assigned U.S. provisional patent application Ser. No.62/072,094, which provisional patent application is hereby incorporatedherein by reference in its entirety to the extent that it does notconflict with the present disclosure.

In some embodiment, instead of manually replacing cell culture mediummanually, a reservoir plate as described herein can be fabricated as aperfusion device in which cell culture medium can flow across thereservoir above the major surface of the wells.

For example and with reference to FIG. 33, a cell culture apparatus asdescribed in and discussed with regard to FIG. 31 4100 can be adaptedsuch that one or more sidewall forms an inlet 4140 and one or moresidewalls forms an outlet 4145. Cell culture fluid can be perfusedacross the reservoir from the inlet to the outlet. The form factor ofthe apparatus depicted in FIG. 33 can be an open top form factor or canbe closed top. If the form factor is open top, an insert including aframe and mesh as discussed with regard to FIG. 2A and FIG. 2B can beused to retain cells within wells 4115 if high perfusion rates thatmight otherwise dislodge cells, such as spheroids, from the wells.

A cell culture apparatus as described herein can be manufactured in anysuitable manner. In various embodiments, a method of manufacturing acell apparatus includes molding a polymeric material, or any othersuitable material as described herein, to form the cell cultureapparatus. The polymeric material may define a plurality of wells of thecell culture apparatus. Each of the plurality of wells may define a topaperture, a bottom surface, and a sidewall surface extending from thetop aperture to the bottom surface. The sidewall surface may also definea pen tip area between the top aperture and the bottom surface. Thepolymeric material may be poured into a mold having pins that causepolymeric material molded around the pins to have the characteristics ofthe plurality of wells as described herein.

In some embodiments, a polymeric material is overmolded onto a substrateto form the cell culture apparatus. The substrate defines the bottomsurface and the combination of the polymeric material and the substratedefines the plurality of wells. The polymeric material may be pouredinto a mold having pins that cause polymeric material molded around thepins to have the characteristics of the plurality of wells as describedherein.

In some embodiments, regardless of how a cell culture apparatusdescribed herein is manufactured, sidewall surfaces of each of aplurality of wells may be coated with a cell non-adherent material asfurther described herein.

In some embodiments, wells comprise various features that are part of,or appended to, the sidewalls. Such substrate features can be directlyinjection molded, or they can be embossed onto formed substrates.Materials of features can be any polymer, polymer blend, co-polymer,glass, metal, or any other material described herein or understood inthe field.

The devices, wells, sidewalls, well-bottom, and other features describedherein are formed of any suitable material. Preferably, materialsintended to contact cells or culture media are compatible with the cellsand the media. Typically, cell culture components are formed frompolymeric material. Examples of suitable polymeric materials includepolystyrene, polymethylmethacrylate, polyvinyl chloride, polycarbonate,polysulfone, polystyrene copolymers, fluoropolymers, polyesters,polyamides, polystyrene butadiene copolymers, fully hydrogenatedstyrenic polymers, polycarbonate PDMS copolymers, and polyolefins suchas polyethylene, polypropylene, polymethyl pentene, polypropylenecopolymers and cyclic olefin copolymers, and the like.

In embodiments, the inner surface of the wells is non-adherent to cells.The wells may be formed from non-adherent material or may be coated withnon-adherent material to form a non-adherent surface. Examplenon-adherent materials include perfluorinated polymers, olefins, or likepolymers or mixtures thereof. Other examples include agarose, non-ionichydrogels such as polyacrylamides, polyethers such as polyethyleneoxide, and polyols such as polyvinyl alcohol, or like materials ormixtures thereof. In some embodiments, the combination of, for example,two or more of non-adherent wells, well geometry, and/or gravity inducescells cultured in the wells to self-assembly into spheroids. Somespheroids maintain differentiated cell function indicative of a more invivo like response relative to cells grown in a monolayer. Inembodiments where the wells are non-adherent to cells, the cells may beharvested by inverting the apparatus to allow gravity to displace thecells from the wells.

In some embodiments, surface modification of materials is used toachieve desired properties. Such modifications include modification ofsurface chemistry and mechanical properties may utilize using biologicalcoatings (e.g., Matrigel™, collagen, laminin, etc.) and syntheticcoatings (e.g., Synthemax®, silicone hydrogels, etc.). Other surfacemodifications to materials (e.g., within wells or microstrucutres) iswithin the scope herein.

A substrate having a structured surface as described herein can beassembled into a cell culture chamber or tray in any suitable manner.For example, the structured surface and one or more other components ofthe cell culture chamber or tray may be molded as a single part. In someembodiments, the structured surface or a portion thereof overmolded toform the bottom and one or more components, the structured surface iswelded (e.g., thermal, laser, long IR or ultrasonic welding, or thelike), adhered, solvent-bonded or the like to one or more othercomponents of the cell culture apparatus.

In various embodiments, a cell culture system can include more than onecell culture apparatus component described herein above. By way ofexample, the apparatus components can be stacked to form a cell culturesystem. Examples of stacked cell culture systems that can incorporate acell culture apparatus component as described herein include thosedescribed in for example, (i) U.S. provisional patent application No.62/072,015, filed on 29 Oct. 2014, entitled “MULTILAYER CULTUREVESSEL,”; (ii) U.S. provisional patent application No. 62/072,039,entitled “PERFUSION BIOREACTOR PLATFORM”, filed on 29 Oct. 2014, whichprovisional patent applications are each hereby incorporated herein byreference in their respective entireties to the extent that they do notconflict with the present disclosure.

The cell culture apparatuses described herein can be used to culturecells within wells of the apparatus in any suitable manner. For example,a method for culturing cells involves introducing cells and a cellculture medium into one or more of the plurality of wells of a cellculture apparatus as described herein. The cell culture medium may becontained in only the cell culture volume or the entirety of each of theplurality of wells including the cell culture volume and the secondvolume. The method also involves culturing cells in the medium in theone or more plurality of wells. Culturing the cells in the one or moreof the plurality of wells may include forming a spheroid within the oneor more wells. The spheroid cultured within the one or more wells may bedefined by a diameter of about, e.g., less than or equal to 500micrometers, less than or equal to 400 micrometers, less than or equalto 300 micrometers, less than or equal to 250 micrometers, less than orequal to 150 micrometers, etc. or any range within the aforementionedvalues The diameter of one spheroid may differ from an average diameterof all the spheroids grown in the plurality of wells by about, e.g.,less than or equal to 20%, less than or equal to 15%, less than or equalto 10%, less than or equal to 5%, less than or equal to 2%, etc. or anyrange within the aforementioned values

In some embodiments, the well-bottom comprises a concave arcuate surfaceor “cup” geometry, for example, a hemi-spherical surface, a conicalsurface having a rounded bottom, and like surface geometries, or acombination thereof. The well (e.g., microwell) and well-bottomultimately terminates, ends, or bottoms-out in a spheroid “friendly”rounded or curved surface, such as a dimple, concave frusto-conicialrelief surfaces, or combinations thereof

In certain embodiments, portions of the sidewalls and/or well-bottom areof varying degrees of opaqueness/transparency to wavelengths within thevisible and/or UV spectrum. For example, opaque sidewall may be combinedwith a transparent microwell-bottom. The transition from opaque totransparent portions may be gradual or immediate.

In some embodiments, the wells (e.g., microwells) comprise alow-adhesion, no-adhesion, or high adhesion coating on a portion of thewell, such as on the at least one concave arcuate surface.

In some embodiments, the device further comprises, for example, a wellannex, well extension area, or an auxiliary side chamber, for receivinga pipette tip for aspiration. In some embodiments, the well annex orwell extension (e.g., a side pocket) is, for example, an integralsurface adjacent to and in fluid communication with the well (e.g.,microwell). In some embodiments, the well annex has a bottom spaced awayfrom a gas-permeable, transparent bottom of the well. The well annex andthe second bottom of the chamber well are, for example spaced away fromthe gas-permeable, transparent bottom such as at a higher elevation orrelative altitude. In some embodiments, the second bottom of the wellannex deflects fluid dispensed from a pipette away from the transparentbottom to avoid disrupting or disturbing the spheroid.

In certain embodiments, the device further comprises a porous membrane,such as a liner or membrane insert, situated within a portion of thewell, situated within a portion of a well annex, or both the well andthe well annex portion. The porous membrane can provide isolation orseparation of a second cellular material, such as a different cell typeor different cell state, situated in an upper portion of the well, in anupper portion of the well formed by the porous membrane, or both wells,from first cellular material in a lower portion of one or both wellsnear bottom.

Devices and well geometries are manufactured by any suitable techniquesknown in the field. In some embodiments, hot embossing, thermaldeformation, and/or injection molding methods are used for theproduction of micropatterned surfaces in cell-culture-compatibleplastics. FIG. 12 depicts schematics of a hot embossing/thermoformingfabrication process that finds use herein. In some embodiments,polystyrene film (or another suitable polymer film) of specificthickness is placed on heat a resistive silicone support. Then the moldis placed on the film with microposts facing down. The whole assembly ispressed under 5N load between plates which are preheated to 130° C. for10 min. After 10 min. plates are cooled below 100° C. and micropatternedembossed/thermoformed film is removed from the assembly and incorporatedinto regular cell culture vessels as a 3D aggregate promoting surface.Other temperatures, times, pressures, and materials may be within thescope herein.

To prevent cell attachment, in some embodiments, micropatterned surfacesare treated with polymers that inhibit cell attachment such aspoly-HEMA, pluronic, or proprietary ULA treatment. Depending on theinitial polymer film thickness and process parameters, surfaces withmicrowells that have different bottom thickness are generated. In someembodiments, polymer thickness of the microwell bottom has a directimpact on oxygen permeability. Thinner microwell bottoms allow betteroxygen supply to cells located inside the microwells. The abovefabrication method delivers a surface with highly oxygen permeablemicrowells.

In some embodiments, devices and systems herein comprise microfluidicelements for the movement of fluids (e.g., media) and cells (e.g.,spheroids) into and out of various compartments, wells, etc. in suchdevices. Microfluidic elements may include channels, reservoirs, valves,pumps, etc.

In vitro 3D tumor cell cultures reflect more accurately complex in vivomicroenvironment than simple two-dimensional cell monolayers. In someembodiments, a cell culture format with microwell patterned surface, asdescribed herein, provides for generating 3D cultures (e.g., tumorspheroids) in large quantities, of uniform size that are compatible withroutine high-throughput drug development and preclinical studies.

In some embodiments, culture vessels described herein find use in theformation of embryoid bodies (EBs) from induced pluripotent stem (iPS)cells and embryonic stem cells (ESCs), allowing for uniform and easyaggregate formation on a large scale. In some embodiments, media changesare performed such that EBs are grown continuously for weeks. In someembodiments, the size of each aggregate is controlled as size isdependent on the number of cells seeded and time of culture. In someembodiments, aggregates are transferred to a traditional well plateallowing for a greater volume of media or analysis of spheroids. In someembodiments, large numbers of formed EB's provide statisticallyimportant data from high throughput analysis of transfected targets orsmall molecules in a single plate. Ability to support formation of largenumber of 3D cell aggregates in one culture vessel makes these vessels,for example modified petri dishes, applicable for the selection ofEB-forming clones for cell reprogramming. In some embodiments, vesselsdescribed herein also support formation of 3D cell aggregates in avariety of cell types related to toxicology, such as hepatocytes andembryonic stem cells. In some embodiments, microwell surface vessels areused for 3D aggregates cell culture with the purpose of, for example,protein production in the bioprocessing field. In some embodiments, acell culture format with microwell patterned surface, as describedherein, provides a means to enable stem cell niche co-culture, inparticular, clonogenic culture, single stem cells and niche co-culture.Combining with staining protocol established for stem cell surfacemarkers or stem cell differentiation markers as well as imaging,computational identification of single stem cells and niche co-culturecan be performed.

Micropatterned vessels are also used for cell banking purposes topreserve cells in 3D format. In some embodiments, once the spheroids aregrown to the specified size (typically at transitional state beforestable state), they are dislodged from the microwells, and the collectedspheroids are cryopreserved and banked for use at a later date.Transitional spheroids mean that the spheroids can continuously grow insize, while the stable spheroids means that the spheroids stop growingin size once reaching its intrinsic size limit. Various cryopreservationmethods are within the scope herein, including, but not limited todimethyl sulfoxide (DMSO), fetal bovine serum (FBS), etc.

EXAMPLES Example 1: Substrate Manufacture

Substrates according to embodiments were manufactured using an embossingmethod, as illustrated in FIG. 12. FIG. 12 shows a hotembossing/thermoforming process of microwell formation in polymer film.Hot plates 1 are provided. Hot plates were preheated to 130° C. Anembossing mold 2 was provided, reflecting the desired well profile. Alayer of polymer film 3 was provided, and a silicone mat 4 was providedbehind the polymer film. The hot plates were heated, and were pressedagainst the polymer film, backed by the silicone layer. When the hotplates were removed, a polymer film having an array of microwells havingthe desired well profile embossed therein was provided.

Example 2: Cell Culture

Experiments conducted during development of embodiments described hereindemonstrate, for example, that 3D cell culture aggregates of uniformdiameter are formed and cultured in an array of individual, spatiallyseparated microwells each having hemispherical round bottoms and roundedtops, with a diameter (D) that is about 1 to 3 times of the desireddiameter of 3D cellular aggregate. The microwell height (H) is equal toabout 0.7 to 1.3 times of the diameter of round bottom part, and thediameter of upper opening of the microwell (D_(top)) is equal to about1.5 to 2.5 times of the round bottom diameter.

Experiments were conducted in which a T25 cell culture flask prototypewith a microwell patterned surface was fabricated and tested in cellculture applications to verify uniformity of spheroid formation andretention/harvesting benefits of proposed design. FIG. 8 contains animage of reverse replica of T25 prototype microwell cross-resectionswith round bottom geometry. Images of spheroids formed by HT29 cells inprototype T25 flask are shown in FIG. 10A. FIG. 10B depicts spheroidsharvested from the flask, which can be compared to those grown usingwith commercially available NUNCLON SPHERA™ low binding surface flask,available from Nunc Nunc/Thermo Fisher, which are depicted in FIG. 11Aand FIG. 11B, the superior performance of the wells described herein isevident in the uniformity of the spheroids produced in embodimentscompared to the commercially available control, which has a low bindingsurface but no wells to define and control spheroid production. Theround bottom geometry is a spheroid inducing geometry. Experiments wereconducted during development of embodiments herein to demonstrate theimpact of oxygen permeability of microwells on cell culture performance.6 and 12 well plates were manufactured, as described above in Example 1,having microwells of different thicknesses. FIG. 13 shows a graphdemonstrating viable cell counts measured after growing cells in 6 wellplates having substrates containing an array of microwells (as describedin Example 1), inmicrowells with different bottom thicknesses. As shownin FIG. 13, A is 70 μm thickness, B is 120 μm thickness and C is 320 μm.Controls was TCT-treated 1 mm thick flat polystyrene. As can be seenfrom the results presented in FIG. 13, thinner material (which exhibitsmore gas permeability) supported more robust cell growth, inembodiments.

FIG. 13 shows a graph demonstrating viable cell counts measured aftergrowing cells in 6 well plates having substrates containing an array ofmicrowells (as described in Example 1), inmicrowells with differentbottom thickness. FIG. 14A and FIG. 14B show graphs comparing viablecell count and cell productivity for substrates having arrays ofmicrowells versus flat surfaces. FIG. 15 shows a graph depicting totalprotein titer excreted from MH677 cells cultured on substrates havingarrays of microwells versus flat surface.

Culture of MH677 cells was performed for the duration of 7 or 9 dayswith media exchange every 2 days. Results presented in FIG. 13demonstrate dependence of total viable cell counts on microwell bottomthickness. Cells cultured in oxygen permeable microwells (column A, 70um thick bottom) yielded an 82% higher viable cell count in comparisonto less oxygen permeable column C, 320 um thick bottom. Overall, cultureon a microwell patterned surface yields higher a viable cell count (FIG.14A) and higher productivity per cell (FIG. 14B) in comparison toregular flat non-adherent surfaces (FIG. 14A and FIG. 14B). Thisproduces 85% higher protein yield (FIG. 15).

Experiments were conducted during development of embodiments herein todemonstrate the advantageous growth of cells in 3D culture vs. 2Dculture using cell culture devices and wells described herein. In bothCHO 5/9 alpha cells (FIG. 34A) and BHK-21 cells (FIG. 34B), vastly moreprotein (hm-CSF in FIG. 34A; EPO in FIG. 34B) was produced per cm² in 3Dculture when compared to 2D.

All publications and patents mentioned in the present application and/orlisted below are herein incorporated by reference. Various modification,recombination, and variation of the described features and embodimentswill be apparent to those skilled in the art without departing from thescope and spirit of the invention. Although specific embodiments havebeen described, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes and embodiments that areobvious to those skilled in the relevant fields are intended to bewithin the scope of the following claims.

REFERENCES

-   1. H. Dolznig, A. Walzl, Organotypic spheroid cultures to study    tumor-stroma interactions during cancer development. Drug discovery    today, V 8., No 2-3, 2011, 113-118-   2. J. Engelberg, G. Ropella, Essential operating principles for    tumor spheroid grwth. BMC Systems Biology 2008, 2, 110-   3. Y. Tung, A. Hsiao, S. Alen. High-throughput 3D spheroid culture    and drug testing using 384 hanging drop array. Analyst, 2011, 136,    473-478-   4. J. Friedrich, C. Seidel, R. Ebner. Spheroid—based drug screen:    considerations and practical approach. Nature protocols, 2009, vol4    no3, 309-323-   5. F. Hirschhaeuser, H. Menne, C. Dittfeld, Multicellular tumor    spheroids: An underestimated tool is catching up again. Journal of    Biotechnology, 2010, 148, 3-15-   6. T. Bartosh, J. Ylostalo, A. Mohammadipoor. Aggregation of human    mesenchymal stromal cells into 3D spheroids enhances their    antiinflammatory properties. PNAS, 2010, 107, no 31, 13724-13729-   7. J. Frith, M. Res, B. Thomson. Dynamic three-dimensional culture    methods enhance mesenchymal stem cell properties and increase    therapeutic potential. Tissue engineering, 2010, 16, no 4, 735-749-   8. S. Sart, A.Tsai, Y. Li. Three-dimensional aggregates of    mesenchymal stem cells: cellular mechanisms, biological properties    and applications. Tissue engineering, 2013, Part B, 00, no 00, 1-16

What is claimed is:
 1. A cell culture substrate comprising an array ofmicrowells, each microwell comprising an opening into the microwelldefined by a rounded top well edge, at least one microwell side-wall,and a rounded well-bottom.
 2. The cell culture substrate of claim 1wherein each microwell, in cross-section, has a sinusoidal well shape.3. The cell culture substrate of claim 1 further comprising one or moreridges, wherein the ridge is round, angular, needle-shaped or hexagonal.4. The cell culture substrate of claim 1 further comprising one or morefissures, wherein the fissure is round, angular, needle-shaped orhexagonal.
 5. The cell culture substrate of claim 1 wherein the roundedwell bottom has a thickness of between 10 and 100 μm.
 6. The cellculture substrate of claim 1 wherein walls of the microwells arerelatively thicker proximate to the opening into the microwell andrelatively thinner at the bottom of the microwell.
 7. The cell culturesubstrate of claim 1 wherein the substrate comprises 2-10000 of saidmicrowells per square centimeter of surface of said cell culturesubstrate.
 8. The cell culture substrate of claim 1 wherein at least therounded well-bottom comprises a non-adherent surface.
 9. The cellculture substrate of claim 1 wherein at least the rounded well-bottomcomprises gas permeable material.
 10. The cell culture substrate ofclaim 1 wherein the microwell sidewalls are discontinuous.
 11. The cellculture substrate of claim 10 wherein the microwell sidewalls arecorrugated.
 12. The cell culture substrate of claim 1 wherein the cellculture substrate comprises at least a portion of a cell culturecontainer.
 13. The cell culture substrate of claim 12 wherein the cellculture container is selected from the group consisting of a multiwellplate, a dish, a flask, a tube, a multi-layer flask, a soft-sided flaskand a bag.
 14. The cell culture substrate of claim 1, wherein microwellsare configured to allow fluid communication between at least one of saidmicrowells and a single liquid reservoir.
 15. The cell culture substrateof claim 12 wherein the cell culture container further comprises a meshplaced on top of the array of microwells.
 16. The cell culture substrateof claim 13 wherein the cell culture container further comprises a meshplaced on top of the array of microwells.
 17. The cell culture substrateof claim 1 wherein the opening into the microwell has an effectivediameter D_(top), the rounded well-bottom has a nadir, and eachmicrowell has one or more sidewalls; the one or more sidewalls have aheight H extending from the nadir of the rounded well-bottom to theopening into the microwell, wherein each microwell has an effectivediameter halfway between the opening into the microwell and the nadir ofthe rounded well-bottom of D_(half-way), and wherein the ratio ofD_(top):D_(half-way) is 1.5:2.5.
 18. The cell culture substrate of claim17 wherein D_(top) is from 200 μm to 500 μm.
 19. The cell culturesubstrate of claim 17 wherein height H is from 100 μm to 500 μm.
 20. Thecell culture substrate of claim 17 wherein H=0.7 to 1.3 D_(half-way).21. The cell culture substrate of claim 17, wherein D_(half-way) is 200to 1000 μm.
 22. A method of culturing spheroids comprising: charging thecell culture container of claim 10 with culture media and spheroidforming cells.